U.S. patent number 7,482,117 [Application Number 10/741,600] was granted by the patent office on 2009-01-27 for genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof.
This patent grant is currently assigned to Celera Corporation. Invention is credited to Michele Cargill, James J. Devlin, Olga Iakoubova.
United States Patent |
7,482,117 |
Cargill , et al. |
January 27, 2009 |
Genetic polymorphisms associated with myocardial infarction,
methods of detection and uses thereof
Abstract
The present invention is based on the discovery of genetic
polymorphisms that are associated with myocardial infarction. In
particular, the present invention relates to nucleic acid molecules
containing the polymorphisms, variant proteins encoded by such
nucleic acid molecules, reagents for detecting the polymorphic
nucleic acid molecules and proteins, and methods of using the
nucleic acid and proteins as well as methods of using reagents for
their detection.
Inventors: |
Cargill; Michele (San
Francisco, CA), Devlin; James J. (Lafayette, CA),
Iakoubova; Olga (Pleasanton, CA) |
Assignee: |
Celera Corporation (Alameda,
CA)
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Family
ID: |
32686282 |
Appl.
No.: |
10/741,600 |
Filed: |
December 22, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050026169 A1 |
Feb 3, 2005 |
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Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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60434778 |
Dec 20, 2002 |
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60453135 |
Mar 10, 2003 |
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60466412 |
Apr 30, 2003 |
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60504955 |
Sep 23, 2003 |
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Current U.S.
Class: |
435/6.14;
536/23.5; 536/24.31; 435/91.2 |
Current CPC
Class: |
A61P
9/10 (20180101); C12Q 1/6883 (20130101); C12Q
2600/156 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 19/34 (20060101); C07H
21/04 (20060101) |
Other References
Larsson, Marten et al. Selective interaction of megalin with
postysynaptic density 95 like membrane associated gyaylated kinase
proteins. 2003. Biochemistry Journal. vol. 373. pp. 381-391. cited
by examiner .
Lucentini, Jack. Gene Association Studies Typically Wrong. 2004.
The Scientist vol. 18, pp. 1-4. cited by examiner .
Wacholder, Sholom et al. Assessing the probability that a positive
report is false: an approach for molecular epidemiology studies.
Journal of the National Cancer Institute. 2004. vol. 96 pp.
434-442. cited by examiner.
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Primary Examiner: Myers; Carla
Assistant Examiner: Shaw; Amanda
Attorney, Agent or Firm: Lee; Victor K. Wang; Ben Karjala;
Justin D.
Claims
What is claimed is:
1. A method of identifying a human having an increased risk for
developing recurrent myocardial infarction, comprising testing a
nucleic acid sample from said human for the presence or absence of
a single nucleotide polymorphism (SNP) at position 101 of SEQ ID
NO: 21398 or its complement thereof, wherein the presence of at
least one G allele at the SNP identifies said human as having an
increased risk for recurrent myocardial infarction as compared to a
human with two A alleles at the SNP.
2. The method of claim 1 in which SEQ ID NO: 21398 is a segment of
the genomic sequence of LRP2 gene comprising SEQ ID NO: 17581.
3. The method of claim 2 in which the SNP is located at position
208593 of SEQ ID NO: 17581.
4. The method of claim 1 in which said nucleic acid sample is
extracted from a biological sample.
5. The method of claim 4 in which said biological sample is blood,
saliva, or buccal cells.
6. The method of claim 1 in which said nucleic acid sample is
amplified before the testing is carried out.
7. The method of claim 1 in which the testing is carried out by
using detection reagents comprising the nucleotide sequences of SEQ
ID NO: 73293, SEQ ID NO: 73294, and SEQ ID NO: 73295.
8. The method of claim 1 in which the testing is carried out by a
process selected from the group consisting of: allele-specific
probe hybridization, allele-specific primer extension,
allele-specific amplification, sequencing, 5' nuclease digestion,
molecular beacon assay, oligonucleotide ligation assay, size
analysis, and single-stranded conformation polymorphism.
9. A method of identifying a human having a decreased risk for
developing recurrent myocardial infarction, comprising testing a
nucleic acid sample from said human for the presence or absence of
a single nucleotide polymorphism (SNP) at position 101 of SEQ ID
NO: 21398 or its complement thereof, wherein the presence of two A
alleles at the SNP identifies said human as having a decreased risk
for recurrent myocardial infarction as compared to a human with at
least one G allele at the SNP.
10. The method of claim 9 in which SEQ ID NO: 21398 is a segment of
the genomic sequence of LRP2 gene comprising SEQ ID NO: 17581.
11. The method of claim 10 in which the SNP is located at position
208593 of SEQ ID NO: 17581.
12. The method of claim 9 in which said nucleic acid sample is
extracted from a biological sample.
13. The method of claim 12 in which said biological sample is
blood, saliva, or buccal cells.
14. The method of claim 9 in which said nucleic acid sample is
amplified before the testing is carried out.
15. The method of claim 9 in which the testing is carried out by
using detection reagents comprising the nucleotide sequences of SEQ
ID NO: 73293, SEQ ID NO: 73294, and SEQ ID NO: 73295.
16. The method of claim 9 in which the testing is carried out by a
process selected from the group consisting of: allele-specific
probe hybridization, allele- specific primer extension,
allele-specific amplification, sequencing, 5' nuclease digestion,
molecular beacon assay, oligonucleotide ligation assay, size
analysis, and single-stranded conformation polymorphism.
17. A method of determining a human's risk for developing recurrent
myocardial infarction, comprising testing a nucleic acid sample
from said human for the presence or absence of a single nucleotide
polymorphism (SNP) at position 101 of SEQ ID NO: 21398 or its
complement thereof, wherein the presence of at least one G allele
at the SNP identifies said human as having an increased risk for
recurrent myocardial infarction as compared to a human with two A
alleles at the SNP, or the presence of two A alleles at the SNP
identifies said human as having a decreased risk for recurrent
myocardial infarction as compared to a human with at least one G
allele at the SNP.
18. The method of claim 17 in which SEQ ID NO: 21398 is a segment
of the genomic sequence of LRP2 gene comprising SEQ ID NO:
17581.
19. The method of claim 18 in which the SNP is located at position
208593 of SEQ ID NO: 17581.
20. The method of claim 17 in which said nucleic acid sample is
extracted from a biological sample.
21. The method of claim 20 in which said biological sample is
blood, saliva, or buccal cells.
22. The method of claim 17 in which said nucleic acid sample is
amplified before the testing is carried out.
23. The method of claim 17 in which the testing is carried out by
using detection reagents comprising the nucleotide sequences of SEQ
ID NO: 73293, SEQ ID NO: 73294, and SEQ ID NO: 73295.
24. The method of claim 17 in which the testing is carried out by a
process selected from the group consisting of: allele-specific
probe hybridization, allele-specific primer extension,
allele-specific amplification, sequencing, 5' nuclease digestion,
molecular beacon assay, oligonucleotide ligation assay, size
analysis, and single-stranded conformation polymorphism.
Description
FIELD OF THE INVENTION
The present invention is in the field of myocardial infarction
diagnosis and therapy. In particular, the present invention relates
to specific single nucleotide polymorphisms (SNPs) in the human
genome, and their association with myocardial infarction (including
recurrent myocardial infarction) and related pathologies. Based on
differences in allele frequencies in the myocardial infarction
patient population relative to normal individuals, the
naturally-occurring SNPs disclosed herein can be used as targets
for the design of diagnostic reagents and the development of
therapeutic agents, as well as for disease association and linkage
analysis. In particular, the SNPs of the present invention are
useful for identifying an individual who is at an increased or
decreased risk of developing myocardial infarction and for early
detection of the disease, for providing clinically important
information for the prevention and/or treatment of myocardial
infarction, and for screening and selecting therapeutic agents. The
SNPs disclosed herein are also useful for human identification
applications. Methods, assays, kits, and reagents for detecting the
presence of these polymorphisms and their encoded products are
provided.
BACKGROUND OF THE INVENTION
Myocardial Infarction (including Recurrent Myocardial
Infarction)
Myocardial infarction (MI) is the most common cause of mortality in
developed countries. It is a multifactorial disease that involves
atherogenesis, thrombus formation and propagation. Thrombosis can
result in complete or partial occlusion of coronary arteries. The
luminal narrowing or blockage of coronary arteries reduces oxygen
and nutrient supply to the cardiac muscle (cardiac ischemia),
leading to myocardial necrosis and/or stunning. MI, unstable
angina, or sudden ischemic death are clinical manifestations of
cardiac muscle damage. All three endpoints are part of the Acute
Coronary Syndrome since the underlying mechanisms of acute
complications of atherosclerosis are considered to be the same.
Atherogenesis, the first step of pathogenesis of MI, is a complex
interaction between blood elements, mechanical forces, disturbed
blood flow, and vessel wall abnormality. On the cellular level,
these include endothelial dysfunction, monocytes/macrophages
activation by modified lipoproteins, monocytes/macrophages
migration into the neointima and subsequent migration and
proliferation of vascular smooth muscle cells (VSMC) from the media
that results in plaque accumulation.
In recent years, an unstable (vulnerable) plaque was recognized as
an underlying cause of arterial thrombotic events and MI. A
vulnerable plaque is a plaque, often not stenotic, that has a high
likelihood of becoming disrupted or eroded, thus forming a
thrombogenic focus. Two vulnerable plaque morphologies have been
described. A first type of vulnerable plaque morphology is a
rupture of the protective fibrous cap. It can occur in plaques that
have distinct morphological features such as large and soft lipid
pool with distinct necrotic core and thinning of the fibrous cap in
the region of the plaque shoulders. Fibrous caps have considerable
metabolic activity. The imbalance between matrix synthesis and
matrix degradation thought to be regulated by inflammatory
mediators combined with VSMC apoptosis are the key underlying
mechanisms of plaque rupture. A second type of vulnerable plaque
morphology, known as "plaque erosion", can also lead to a fatal
coronary thrombotic event. Plaque erosion is morphologically
different from plaque rupture. Eroded plaques do not have fractures
in the plaque fibrous cap, only superficial erosion of the intima.
The loss of endothelial cells can expose the thrombogenic
subendothelial matrix that precipitates thrombus formation. This
process could be regulated by inflammatory mediators. The
propagation of the acute thrombi for both plaque rupture and plaque
erosion events depends on the balance between coagulation and
thrombolysis. MI due to a vulnerable plaque is a complex phenomenon
that includes: plaque vulnerability, blood vulnerability
(hypercoagulation, hypothrombolysis), and heart vulnerability
(sensitivity of the heart to ischemia or propensity for
arrhythmia).
Recurrent myocardial infarction can generally be viewed as a severe
form of MI progression caused by multiple vulnerable plaques that
are able to undergo pre-rupture or a pre-erosive state, coupled
with extreme blood coagulability.
The incidence of MI is still high despite currently available
preventive measures and therapeutic intervention. More than
1,500,000 people in the US suffer acute MI each year (many without
seeking help due to unrecognized MI), and one third of these people
die. The lifetime risk of coronary artery disease events at age 40
years is 42.4% for men (one in two) and 24.9% for women (one in
four) (Lloyd-Jones D M; Lancet, 1999 353: 89-92).
The current diagnosis of MI is based on the levels of troponin I or
T that indicate the cardiac muscle progressive necrosis, impaired
electrocardiogram (ECG), and detection of abnormal ventricular wall
motion or angiographic data (the presence of acute thrombi).
However, due to the asymptomatic nature of 25% of acute MIs
(absence of atypical chest pain, low ECG sensitivity), a
significant portion of MIs are not diagnosed and therefore not
treated appropriately (e.g., prevention of recurrent MIs).
Despite a very high prevalence and lifetime risk of MI, there are
no good prognostic markers that can identify an individual with a
high risk of vulnerable plaques and justify preventive treatments.
MI risk assessment and prognosis is currently done using classic
risk factors or the recently introduced Framingham Risk Index. Both
of these assessments put a significant weight on LDL levels to
justify preventive treatment. However, it is well established that
half of all MIs occur in individuals without overt hyperlipidemia.
Hence, there is a need for additional risk factors for predicting
predisposition to MI.
Other emerging risk factors are inflammatory biomarkers such as
C-reactive protein (CRP), ICAM-1, SAA, TNF .alpha., homocysteine,
impaired fasting glucose, new lipid markers (ox LDL, Lp-a, MAD-LDL,
etc.) and pro-thrombotic factors (fibrinogen, PAI-1). Despite
showing some promise, these markers have significant limitations
such as low specificity and low positive predictive value, and the
need for multiple reference intervals to be used for different
groups of people (e.g., males-females, smokers-non smokers, hormone
replacement therapy users, different age groups). These limitations
diminish the utility of such markers as independent prognostic
markers for MI screening.
Genetics plays an important role in MI risk. Families with a
positive family history of MI account for 14% of the general
population, 72% of premature MIs, and 48% of all MIs (Williams R R,
Am J Cardiology, 2001; 87:129). In addition, replicated linkage
studies have revealed evidence of multiple regions of the genome
that are associated with MI and relevant to MI genetic traits,
including regions on chromosomes 14, 2, 3 and 7 (Broeckel U, Nature
Genetics, 2002; 30: 210; Harrap S, Arterioscler Thromb Vasc Biol,
2002; 22: 874-878, Shearman A, Human Molecular Genetics, 2000, 9;
9, 1315-1320), implying that genetic risk factors influence the
onset, manifestation, and progression of MI. Recent association
studies have identified allelic variants that are associated with
acute complications of coronary heart disease, including allelic
variants of the ApoE, ApoA5, Lpa, APOCIII, and Klotho genes.
Genetic markers such as single nucleotide polymorphisms are
preferable to other types of biomarkers. Genetic markers that are
prognostic for MI can be genotyped early in life and could predict
individual response to various risk factors. The combination of
serum protein levels and genetic predisposition revealed by genetic
analysis of susceptibility genes can provide an integrated
assessment of the interaction between genotypes and environmental
factors, resulting in synergistically increased prognostic value of
diagnostic tests.
Thus, there is an urgent need for novel genetic markers that are
predictive of predisposition to MI, particularly for individuals
who are unrecognized as having a predisposition to MI. Such genetic
markers may enable prognosis of MI in much larger populations
compared with the populations which can currently be evaluated by
using existing risk factors and biomarkers. The availability of a
genetic test may allow, for example, appropriate preventive
treatments for acute coronary events to be provided for susceptible
individuals (such preventive treatments may include, for example,
statin treatments and statin dose escalation, as well as changes to
modifiable risk factors), lowering of the thresholds for ECG and
angiography testing, and allow adequate monitoring of informative
biomarkers.
Moreover, the discovery of genetic markers associated with MI will
provide novel targets for therapeutic intervention or preventive
treatments of MI, and enable the development of new therapeutic
agents for treating MI and other cardiovascular disorders.
SNPs
The genomes of all organisms undergo spontaneous mutation in the
course of their continuing evolution, generating variant forms of
progenitor genetic sequences (Gusella, Ann. Rev. Biochem. 55,
831-854 (1986)). A variant form may confer an evolutionary
advantage or disadvantage relative to a progenitor form or may be
neutral. In some instances, a variant form confers an evolutionary
advantage to the species and is eventually incorporated into the
DNA of many or most members of the species and effectively becomes
the progenitor form. Additionally, the effects of a variant form
may be both beneficial and detrimental, depending on the
circumstances. For example, a heterozygous sickle cell mutation
confers resistance to malaria, but a homozygous sickle cell
mutation is usually lethal. In many cases, both progenitor and
variant forms survive and co-exist in a species population. The
coexistence of multiple forms of a genetic sequence gives rise to
genetic polymorphisms, including SNPs.
Approximately 90% of all polymorphisms in the human genome are
SNPs. SNPs are single base positions in DNA at which different
alleles, or alternative nucleotides, exist in a population. The SNP
position (interchangeably referred to herein as SNP, SNP site, or
SNP locus) is usually preceded by and followed by highly conserved
sequences of the allele (e.g., sequences that vary in less than
1/100 or 1/1000 members of the populations). An individual may be
homozygous or heterozygous for an allele at each SNP position. A
SNP can, in some instances, be referred to as a "cSNP" to denote
that the nucleotide sequence containing the SNP is an amino acid
coding sequence.
A SNP may arise from a substitution of one nucleotide for another
at the polymorphic site. Substitutions can be transitions or
transversions. A transition is the replacement of one purine
nucleotide by another purine nucleotide, or one pyrimidine by
another pyrimidine. A transversion is the replacement of a purine
by a pyrimidine, or vice versa. A SNP may also be a single base
insertion or deletion variant referred to as an "indel" (Weber et
al., "Human diallelic insertion/deletion polymorphisms", Am J Hum
Genet 2002 October; 71(4):854-62).
A synonymous codon change, or silent mutation/SNP (terms such as
"SNP", "polymorphism", "mutation", "mutant", "variation", and
"variant" are used herein interchangeably), is one that does not
result in a change of amino acid due to the degeneracy of the
genetic code. A substitution that changes a codon coding for one
amino acid to a codon coding for a different amino acid (i.e., a
non-synonymous codon change) is referred to as a missense mutation.
A nonsense mutation results in a type of non-synonymous codon
change in which a stop codon is formed, thereby leading to
premature termination of a polypeptide chain and a truncated
protein. A read-through mutation is another type of non-synonymous
codon change that causes the destruction of a stop codon, thereby
resulting in an extended polypeptide product. While SNPs can be
bi-, tri-, or tetra-allelic, the vast majority of the SNPs are
bi-allelic, and are thus often referred to as "bi-allelic markers",
or "di-allelic markers".
As used herein, references to SNPs and SNP genotypes include
individual SNPs and/or haplotypes, which are groups of SNPs that
are generally inherited together. Haplotypes can have stronger
correlations with diseases or other phenotypic effects compared
with individual SNPs, and therefore may provide increased
diagnostic accuracy in some cases (Stephens et al. Science 293,
489-493, 20 Jul. 2001).
Causative SNPs are those SNPs that produce alterations in gene
expression or in the expression, structure, and/or function of a
gene product, and therefore are most predictive of a possible
clinical phenotype. One such class includes SNPs falling within
regions of genes encoding a polypeptide product, i.e. cSNPs. These
SNPs may result in an alteration of the amino acid sequence of the
polypeptide product (i.e., non-synonymous codon changes) and give
rise to the expression of a defective or other variant protein.
Furthermore, in the case of nonsense mutations, a SNP may lead to
premature termination of a polypeptide product. Such variant
products can result in a pathological condition, e.g., genetic
disease. Examples of genes in which a SNP within a coding sequence
causes a genetic disease include sickle cell anemia and cystic
fibrosis.
Causative SNPs do not necessarily have to occur in coding regions;
causative SNPs can occur in, for example, any genetic region that
can ultimately affect the expression, structure, and/or activity of
the protein encoded by a nucleic acid. Such genetic regions
include, for example, those involved in transcription, such as SNPs
in transcription factor binding domains, SNPs in promoter regions,
in areas involved in transcript processing, such as SNPs at
intron-exon boundaries that may cause defective splicing, or SNPs
in mRNA processing signal sequences such as polyadenylation signal
regions. Some SNPs that are not causative SNPs nevertheless are in
close association with, and therefore segregate with, a
disease-causing sequence. In this situation, the presence of a SNP
correlates with the presence of, or predisposition to, or an
increased risk in developing the disease. These SNPs, although not
causative, are nonetheless also useful for diagnostics, disease
predisposition screening, and other uses.
An association study of a SNP and a specific disorder involves
determining the presence or frequency of the SNP allele in
biological samples from individuals with the disorder of interest,
such as myocardial infarction, and comparing the information to
that of controls (i.e., individuals who do not have the disorder;
controls may be also referred to as "healthy" or "normal"
individuals) who are preferably of similar age and race. The
appropriate selection of patients and controls is important to the
success of SNP association studies. Therefore, a pool of
individuals with well-characterized phenotypes is extremely
desirable.
A SNP may be screened in diseased tissue samples or any biological
sample obtained from a diseased individual, and compared to control
samples, and selected for its increased (or decreased) occurrence
in a specific pathological condition, such as pathologies related
to myocardial infarction. Once a statistically significant
association is established between one or more SNP(s) and a
pathological condition (or other phenotype) of interest, then the
region around the SNP can optionally be thoroughly screened to
identify the causative genetic locus/sequence(s) (e.g., causative
SNP/mutation, gene, regulatory region, etc.) that influences the
pathological condition or phenotype. Association studies may be
conducted within the general population and are not limited to
studies performed on related individuals in affected families
(linkage studies).
Clinical trials have shown that patient response to treatment with
pharmaceuticals is often heterogeneous. There is a continuing need
to improve pharmaceutical agent design and therapy. In that regard,
SNPs can be used to identify patients most suited to therapy with
particular pharmaceutical agents (this is often termed
"pharmacogenomics"). Similarly, SNPs can be used to exclude
patients from certain treatment due to the patient's increased
likelihood of developing toxic side effects or their likelihood of
not responding to the treatment. Pharmacogenomics can also be used
in pharmaceutical research to assist the drug development and
selection process. (Linder et al. (1997), Clinical Chemistry, 43,
254; Marshall (1997), Nature Biotechnology, 15, 1249; International
Patent Application WO 97/40462, Spectra Biomedical; and Schafer et
al. (1998), Nature Biotechnology, 16, 3).
SUMMARY OF THE INVENTION
The present invention relates to the identification of novel SNPs,
unique combinations of such SNPs, and haplotypes of SNPs that are
associated with myocardial infarction (including recurrent
myocardial infarction) and related pathologies. The polymorphisms
disclosed herein are directly useful as targets for the design of
diagnostic reagents and the development of therapeutic agents for
use in the diagnosis and treatment of myocardial infarction and
related pathologies.
Based on the identification of SNPs associated with myocardial
infarction, the present invention also provides methods of
detecting these variants as well as the design and preparation of
detection reagents needed to accomplish this task. The invention
specifically provides novel SNPs in genetic sequences involved in
myocardial infarction, variant proteins encoded by nucleic acid
molecules containing such SNPs, antibodies to the encoded variant
proteins, computer-based and data storage systems containing the
novel SNP information, methods of detecting these SNPs in a test
sample, methods of identifying individuals who have an altered
(i.e., increased or decreased) risk of developing myocardial
infarction based on the presence of a SNP disclosed herein or its
encoded product, methods of identifying individuals who are more or
less likely to respond to a treatment, methods of screening for
compounds useful in the treatment of a disorder associated with a
variant gene/protein, compounds identified by these methods,
methods of treating disorders mediated by a variant gene/protein,
and methods of using the novel SNPs of the present invention for
human identification.
In Tables 1-2, the present invention provides gene information,
transcript sequences (SEQ ID NOS:1-828), encoded amino acid
sequences (SEQ ID NOS:829-1656), genomic sequences (SEQ ID
NOS:17,553-18,016), transcript-based context sequences (SEQ ID
NOS:1657-17,552) and genomic-based context sequences (SEQ ID
NOS:18,017-73,085) that contain the SNPs of the present invention,
and extensive SNP information that includes observed alleles,
allele frequencies, populations/ethnic groups in which alleles have
been observed, information about the type of SNP and corresponding
functional effect, and, for cSNPs, information about the encoded
polypeptide product. The transcript sequences (SEQ ID NOS:1-828),
amino acid sequences (SEQ ID NOS:829-1656), genomic sequences (SEQ
ID NOS:17,553-18,016), transcript-based SNP context sequences (SEQ
ID NOS: 1657-17,552), and genomic-based SNP context sequences (SEQ
ID NOS:18,017-73,085) are also provided in the Sequence
Listing.
In a specific embodiment of the present invention,
naturally-occurring SNPs in the human genome are provided. These
SNPs are associated with myocardial infarction such that they can
have a variety of uses in the diagnosis and/or treatment of
myocardial infarction. One aspect of the present invention relates
to an isolated nucleic acid molecule comprising a nucleotide
sequence in which at least one nucleotide is a SNP disclosed in
Tables 3 and/or 4. In an alternative embodiment, a nucleic acid of
the invention is an amplified polynucleotide, which is produced by
amplification of a SNP-containing nucleic acid template. In another
embodiment, the invention provides for a variant protein which is
encoded by a nucleic acid molecule containing a SNP disclosed
herein.
In yet another embodiment of the invention, a reagent for detecting
a SNP in the context of its naturally-occurring flanking nucleotide
sequences (which can be, e.g., either DNA or mRNA) is provided. In
particular, such a reagent may be in the form of, for example, a
hybridization probe or an amplification primer that is useful in
the specific detection of a SNP of interest. In an alternative
embodiment, a protein detection reagent is used to detect a variant
protein which is encoded by a nucleic acid molecule containing a
SNP disclosed herein. A preferred embodiment of a protein detection
reagent is an antibody or an antigen-reactive antibody
fragment.
Also provided in the invention are kits comprising SNP detection
reagents, and methods for detecting the SNPs disclosed herein by
employing detection reagents. In a specific embodiment, the present
invention provides for a method of identifying an individual having
an increased or decreased risk of developing myocardial infarction
by detecting the presence or absence of a SNP allele disclosed
herein. In another embodiment, a method for diagnosis of myocardial
infarction by detecting the presence or absence of a SNP allele
disclosed herein is provided.
The nucleic acid molecules of the invention can be inserted in an
expression vector, such as to produce a variant protein in a host
cell. Thus, the present invention also provides for a vector
comprising a SNP-containing nucleic acid molecule,
genetically-engineered host cells containing the vector, and
methods for expressing a recombinant variant protein using such
host cells. In another specific embodiment, the host cells,
SNP-containing nucleic acid molecules, and/or variant proteins can
be used as targets in a method for screening and identifying
therapeutic agents or pharmaceutical compounds useful in the
treatment of myocardial infarction.
An aspect of this invention is a method for treating myocardial
infarction in a human subject wherein said human subject harbors a
gene, transcript, and/or encoded protein identified in Tables 1-2,
which method comprises administering to said human subject a
therapeutically or prophylactically effective amount of one or more
agents counteracting the effects of the disease, such as by
inhibiting (or stimulating) the activity of the gene, transcript,
and/or encoded protein identified in Tables 1-2.
Another aspect of this invention is a method for identifying an
agent useful in therapeutically or prophylactically treating
myocardial infarction in a human subject wherein said human subject
harbors a gene, transcript, and/or encoded protein identified in
Tables 1-2, which method comprises contacting the gene, transcript,
or encoded protein with a candidate agent under conditions suitable
to allow formation of a binding complex between the gene,
transcript, or encoded protein and the candidate agent and
detecting the formation of the binding complex, wherein the
presence of the complex identifies said agent.
Another aspect of this invention is a method for treating
myocardial infarction in a human subject, which method
comprises:
(i) determining that said human subject harbors a gene, transcript,
and/or encoded protein identified in Tables 1-2, and
(ii) administering to said subject a therapeutically or
prophylactically effective amount of one or more agents
counteracting the effects of the disease.
Many other uses and advantages of the present invention will be
apparent to those skilled in the art upon review of the detailed
description of the preferred embodiments herein. Solely for clarity
of discussion, the invention is described in the sections below by
way of non-limiting examples.
DESCRIPTION OF THE FILES CONTAINED ON THE CD-R NAMED
CL001499CDR
The CD-R named CL001499CDR contains the following five text (ASCII)
files:
1) File SEQLIST.sub.--1499.txt provides the Sequence Listing. The
Sequence Listing provides the transcript sequences (SEQ ID
NOS:1-828) and protein sequences (SEQ ID NOS:829-1656) as shown in
Table 1, and genomic sequences (SEQ ID NOS:17,553-18,016) as shown
in Table 2, for each myocardial infarction-associated gene that
contains one or more SNPs of the present invention. Also provided
in the Sequence Listing are context sequences flanking each SNP,
including both transcript-based context sequences as shown in Table
1 (SEQ ID NOS:1657-17,552) and genomic-based context sequences as
shown in Table 2 (SEQ ID NOS:18,017-73,085). The context sequences
generally provide 100 bp upstream (5') and 100 bp downstream (3')
of each SNP, with the SNP in the middle of the context sequence,
for a total of 200 bp of context sequence surrounding each SNP.
File SEQLIST.sub.--1499.txt is 65,130 KB in size.
2) File TABLE1.sub.--1499.txt provides Table 1. File
TABLE1.sub.--1499.txt is 13,068 KB in size.
3) File TABLE2.sub.--1499.txt provides Table 2. File
TABLE2.sub.--1499.txt is 52,658 KB in size.
4) File TABLE3.sub.--1499.txt provides Table 3. File
TABLE3.sub.--1499.txt is 120 KB in size.
5) File TABLE4.sub.--1499.txt provides Table 4. File
TABLE4.sub.--1499.txt is 159 KB in size.
The material contained on the CD-R labeled CL001499CDR is hereby
incorporated by reference pursuant to 37 CFR 1.77(b)(4).
Description of Table 1 and Table 2
Table 1 and Table 2 (both provided on the CD-R) disclose the SNP
and associated gene/transcript/protein information of the present
invention. For each gene, Table 1 and Table 2 each provide a header
containing gene/transcript/protein information, followed by a
transcript and protein sequence (in Table 1) or genomic sequence
(in Table 2), and then SNP information regarding each SNP found in
that gene/transcript.
NOTE: SNPs may be included in both Table 1 and Table 2; Table 1
presents the SNPs relative to their transcript sequences and
encoded protein sequences, whereas Table 2 presents the SNPs
relative to their genomic sequences (in some instances Table 2 may
also include, after the last gene sequence, genomic sequences of
one or more intergenic regions, as well as SNP context sequences
and other SNP information for any SNPs that lie within these
intergenic regions). SNPs can readily be cross-referenced between
Tables based on their hCV (or, in some instances, hDV)
identification numbers.
The gene/transcript/protein information includes: a gene number (1
through n, where n=the total number of genes in the Table) a Celera
hCG and UID internal identification numbers for the gene a Celera
hCT and UID internal identification numbers for the transcript
(Table 1 only) a public Genbank accession number (e.g., RefSeq NM
number) for the transcript (Table 1 only) a Celera hCP and UID
internal identification numbers for the protein encoded by the hCT
transcript (Table 1 only) a public Genbank accession number (e.g.,
RefSeq NP number) for the protein (Table 1 only) an art-known gene
symbol an art-known gene/protein name Celera genomic axis position
(indicating start nucleotide position-stop nucleotide position) the
chromosome number of the chromosome on which the gene is located an
OMIM (Online Mendelian Inheritance in Man; Johns Hopkins
University/NCBI) public reference number for obtaining further
information regarding the medical significance of each gene
alternative gene/protein name(s) and/or symbol(s) in the OMIM
entry
NOTE: Due to the presence of alternative splice forms, multiple
transcript/protein entries can be provided for a single gene entry
in Table 1; i.e., for a single Gene Number, multiple entries may be
provided in series that differ in their transcript/protein
information and sequences.
Following the gene/transcript/protein information is a transcript
sequence and protein sequence (in Table 1), or a genomic sequence
(in Table 2), for each gene, as follows: transcript sequence (Table
1 only) (corresponding to SEQ ID NOS:1-828 of the Sequence
Listing), with SNPs identified by their IUB codes (transcript
sequences can include 5' UTR, protein coding, and 3' UTR regions).
(NOTE: If there are differences between the nucleotide sequence of
the hCT transcript and the corresponding public transcript sequence
identified by the Genbank accession number, the hCT transcript
sequence (and encoded protein) is provided, unless the public
sequence is a RefSeq transcript sequence identified by an NM
number, in which case the RefSeq NM transcript sequence (and
encoded protein) is provided. However, whether the hCT transcript
or RefSeq NM transcript is used as the transcript sequence, the
disclosed SNPs are represented by their IUB codes within the
transcript.) the encoded protein sequence (Table 1 only)
(corresponding to SEQ ID NOS:829-1656 of the Sequence Listing) the
genomic sequence of the gene (Table 2 only), including 6 kb on each
side of the gene boundaries (i.e., 6 kb on the 5' side of the gene
plus 6 kb on the 3' side of the gene) (corresponding to SEQ ID
NOS:17,553-18,016 of the Sequence Listing).
After the last gene sequence, Table 2 may include additional
genomic sequences of intergenic regions (in such instances, these
sequences are identified as "Intergenic region:" followed by a
numerical identification number), as well as SNP context sequences
and other SNP information for any SNPs that lie within each
intergenic region (and such SNPs are identified as "INTERGENIC" for
SNP type).
NOTE: The transcript, protein, and transcript-based SNP context
sequences are provided in both Table 1 and in the Sequence Listing.
The genomic and genomic-based SNP context sequences are provided in
both Table 2 and in the Sequence Listing. SEQ ID NOS are indicated
in Table 1 for each transcript sequence (SEQ ID NOS:1-828), protein
sequence (SEQ ID NOS:829-1656), and transcript-based SNP context
sequence (SEQ ID NOS:1657-17,552), and SEQ ID NOS are indicated in
Table 2 for each genomic sequence (SEQ ID NOS:17,553-18,016), and
genomic-based SNP context sequence (SEQ ID NOS:18,017-73,085).
The SNP information includes: context sequence (taken from the
transcript sequence in Table 1, and taken from the genomic sequence
in Table 2) with the SNP represented by its IUB code, including 100
bp upstream (5') of the SNP position plus 100 bp downstream (3') of
the SNP position (the transcript-based SNP context sequences in
Table 1 are provided in the Sequence Listing as SEQ ID
NOS:1657-17,552; the genomic-based SNP context sequences in Table 2
are provided in the Sequence Listing as SEQ ID NOS:18,017-73,085).
Celera hCV internal identification number for the SNP (in some
instances, an "hDV" number is given instead of an "hCV" number) SNP
position [position of the SNP within the given transcript sequence
(Table 1) or within the given genomic sequence (Table 2)] SNP
source (may include any combination of one or more of the following
five codes, depending on which internal sequencing projects and/or
public databases the SNP has been observed in: "Applera"=SNP
observed during the re-sequencing of genes and regulatory regions
of 39 individuals, "Celera"=SNP observed during shotgun sequencing
and assembly of the Celera human genome sequence, "Celera
Diagnostics"=SNP observed during re-sequencing of nucleic acid
samples from individuals who have myocardial infarction or a
related pathology, "dbSNP"=SNP observed in the dbSNP public
database, "HGBASE"=SNP observed in the HGBASE public database,
"HGMD"=SNP observed in the Human Gene Mutation Database (HGMD)
public database) (NOTE: multiple "Applera" source entries for a
single SNP indicate that the same SNP was covered by multiple
overlapping amplification products and the re-sequencing results
(e.g., observed allele counts) from each of these amplification
products is being provided) Population/allele/allele count
information in the format of
[population1(allele1,count|allele2,count)
population2(allele1,count|allele2,count) total (allele1,total
count|allele2,total count)]. The information in this field includes
populations/ethnic groups in which particular SNP alleles have been
observed ("cau"=Caucasian, "his"=Hispanic, "chn"=Chinese, and
"afr"=African-American, "jpn"=Japanese, "ind"=Indian,
"mex"=Mexican, "ain"="American Indian, "cra"=Celera donor,
"no_pop"=no population information available), identified SNP
alleles, and observed allele counts (within each population group
and total allele counts), where available ["-" in the allele field
represents a deletion allele of an insertion/deletion ("indel")
polymorphism (in which case the corresponding insertion allele,
which may be comprised of one or more nucleotides, is indicated in
the allele field on the opposite side of the "|"); "-" in the count
field indicates that allele count information is not
available].
NOTE: For SNPs of "Applera" SNP source, genes/regulatory regions of
39 individuals (20 Caucasians and 19 African Americans) were
re-sequenced and, since each SNP position is represented by two
chromosomes in each individual (with the exception of SNPs on X and
Y chromosomes in males, for which each SNP position is represented
by a single chromosome), up to 78 chromosomes were genotyped for
each SNP position. Thus, the sum of the African-American ("afr")
allele counts is up to 38, the sum of the Caucasian allele counts
("cau") is up to 40, and the total sum of all allele counts is up
to 78.
(NOTE: semicolons separate population/allele/count information
corresponding to each indicated SNP source; i.e., if four SNP
sources are indicated, such as "Celera", "dbSNP", "HGBASE", and
"HGMD", then population/allele/count information is provided in
four groups which are separated by semicolons and listed in the
same order as the listing of SNP sources, with each
population/allele/count information group corresponding to the
respective SNP source based on order; thus, in this example, the
first population/allele/count information group would correspond to
the first listed SNP source (Celera) and the third
population/allele/count information group separated by semicolons
would correspond to the third listed SNP source (HGBASE); if
population/allele/count information is not available for any
particular SNP source, then a pair of semicolons is still inserted
as a place-holder in order to maintain correspondence between the
list of SNP sources and the corresponding listing of
population/allele/count information)
SNP type (e.g., location within gene/transcript and/or predicted
functional effect) ["MIS-SENSE MUTATION"=SNP causes a change in the
encoded amino acid (i.e., a non-synonymous coding SNP); "SILENT
MUTATION"=SNP does not cause a change in the encoded amino acid
(i.e., a synonymous coding SNP); "STOP CODON MUTATION"=SNP is
located in a stop codon; "NONSENSE MUTATION"=SNP creates or
destroys a stop codon; "UTR 5"=SNP is located in a 5' UTR of a
transcript; "UTR 3"=SNP is located in a 3' UTR of a transcript;
"PUTATIVE UTR 5"=SNP is located in a putative 5' UTR; "PUTATIVE UTR
3"=SNP is located in a putative 3' UTR; "DONOR SPLICE SITE"=SNP is
located in a donor splice site (5' intron boundary); "ACCEPTOR
SPLICE SITE"=SNP is located in an acceptor splice site (3' intron
boundary); "CODING REGION"=SNP is located in a protein-coding
region of the transcript; "EXON"=SNP is located in an exon;
"INTRON"=SNP is located in an intron; "hmCS"=SNP is located in a
human-mouse conserved segment; "TFBS"=SNP is located in a
transcription factor binding site; "UNKNOWN"=SNP type is not
defined; "INTERGENIC"=SNP is intergenic, i.e., outside of any gene
boundary]
Protein coding information (Table 1 only), where relevant, in the
format of [protein SEQ ID NO:#, amino acid position, (amino acid-1,
codon1) (amino acid-2, codon2)]. The information in this field
includes SEQ ID NO of the encoded protein sequence, position of the
amino acid residue within the protein identified by the SEQ ID NO
that is encoded by the codon containing the SNP, amino acids
(represented by one-letter amino acid codes) that are encoded by
the alternative SNP alleles (in the case of stop codons, "X" is
used for the one-letter amino acid code), and alternative codons
containing the alternative SNP nucleotides which encode the amino
acid residues (thus, for example, for missense mutation-type SNPs,
at least two different amino acids and at least two different
codons are generally indicated; for silent mutation-type SNPs, one
amino acid and at least two different codons are generally
indicated, etc.). In instances where the SNP is located outside of
a protein-coding region (e.g., in a UTR region), "None" is
indicated following the protein SEQ ID NO.
Description Table 3 and Table 4
Tables 3 and 4 (both provided on the CD-R) provide a list of a
subset of SNPs from Table 1 (in the case of Table 3) or Table 2 (in
the case of Table 4) for which the SNP source falls into one of the
following three categories: 1) SNPs for which the SNP source is
only "Applera" and none other, 2) SNPs for which the SNP source is
only "Celera Diagnostics" and none other, and 3) SNPs for which the
SNP source is both "Applera" and "Celera Diagnostics" but none
other.
These SNPs have not been observed in any of the public databases
(dbSNP, HGBASE, and HGMD), and were also not observed during
shotgun sequencing and assembly of the Celera human genome sequence
(i.e., "Celera" SNP source). Tables 3 and 4 provide the hCV
identification number (or hDV identification number for SNPs having
"Celera Diagnostics" SNP source) and the SEQ ID NO of the context
sequence for each of these SNPs.
Description of Table 5
Table 5 provides sequences (SEQ ID NOS:73,086-73,997) of primers
that have been synthesized and used in the laboratory to carry out
allele-specific PCR reactions in order to assay the SNPs disclosed
in Tables 6-7 during the course of myocardial infarction
association studies.
Table 5 provides the following: the column labeled "hCV" provides
an hCV identification number for each SNP site the column labeled
"Alleles" designates the two alternative alleles at the SNP site
identified by the hCV-identification number that are targeted by
the allele-specific primers (the allele-specific primers are shown
as "Sequence A" and "Sequence B" in each row) the column labeled
"Sequence A (allele-specific primer)" provides an allele-specific
primer that is specific for the first allele designated in the
"Alleles" column the column labeled "Sequence B (allele-specific
primer)" provides an allele-specific primer that is specific for
the second allele designated in the "Alleles" column the column
labeled "Sequence C (common primer)" provides a common primer that
is used in conjunction with each of the allele-specific primers
(the "Sequence A" primer and the "Sequence B" primer) and which
hybridizes at a site away from the SNP position.
All primer sequences are given in the 5' to 3' direction.
Each of the alleles designated in the "Alleles" column matches the
3' nucleotide of the allele-specific primer that is specific for
that allele. Thus, the first allele designated in the "Alleles"
column matches the 3' nucleotide of the "Sequence A" primer, and
the second allele designated in the "Alleles" column matches the 3'
nucleotide of the "Sequence B" primer.
Description of Table 6 and Table 7
Tables 6 and 7 provide results of statistical analyses for SNPs
disclosed in Tables 1-5 (SNPs can be cross-referenced between
Tables based on their hCV identification numbers). Table 6 provides
statistical results for association of SNPs with myocardial
infarction, and Table 7 provides statistical results for
association of SNPs with recurrent myocardial infarction (RMI). The
statistical results shown in Tables 6-7 provide support for the
association of these SNPs with MI (Table 6) and/or RMI (Table 7).
For example, the statistical results provided in Tables 6-7 show
that the association of these SNPs with MI and/or RMI is supported
by p-values <0.05 in at least one of three genotypic association
tests and/or an allelic association test. Moreover, in general, the
SNPs identified in Tables 6-7 are SNPs for which their association
with MI and/or RMI has been replicated by virtue of being
significant in at least two independently collected sample sets,
which further verifies the association of these SNPs with MI and/or
RMI. Furthermore, results of stratification-based analyses are also
provided; stratified analysis can, for example, enable increased
prediction of MI and/or RMI risk via interaction between
conventional risk factors (stratum) and SNPs.
NOTE: SNPs can be cross-referenced between Tables 1-7 based on the
hCV identification number of each SNP. However, 16 of the SNPs that
are included in Tables 1-7 possess two different hCV identification
numbers, as follows: hCV11159941 is equivalent to hCV26841917
hCV1129436 is equivalent to hCV26581155 hCV15751934 is equivalent
to hCV25474315 hCV1597697 is equivalent to hCV22274031 hCV16072719
is equivalent to hCV25473700 hCV16089120 is equivalent to
hCV25473150 hCV16172571 is equivalent to hCV25474627 hCV16192174 is
equivalent to hCV22271999 hCV16273460 is equivalent to hCV26165616
hCV2303890 is equivalent to hCV27861075 hCV25772464 is equivalent
to hCV2531732 hCV7482175 is equivalent to hCV26546221 hCV7499900 is
equivalent to hCV25620145 hCV7591528 is equivalent to hCV25618313
hCV9485713 is equivalent to hCV25640926 hCV9494470 is equivalent to
hCV26665714
TABLE-US-00001 TABLE 6 (SNP association with Myocardial Infarction)
Description of column headings for Table 6: Table 6 column heading
Definition Marker Internal hCV identification number for the SNP
that is tested Study Sample set used in the analysis Stratification
Indicates if the analysis of the dataset was done on a substratum
(stratifications are described below) Strata Indicates what
substratum was used in the analysis (strata are described below)
Status Identifies the inclusion/exclusion criteria for cases and
controls (described below) Allele1 Nucleotide (allele) of the
tested SNP for which statistics are being reported Case Allele1 frq
Allele frequency of Allele1 in cases Control Allele1 frq Allele
frequency of Allele1 in controls Allelic p-value Result of the
Fisher exact test for allelic association Dom p-value Result of the
asymptotic chi square test for dominant genotypic association Rec
p-value Result of the asymptotic chi square test for recessive
genotypic association OR Allelic odds ratio OR 95% CI L Lower limit
of 95% confidence interval of the allelic odds ratio OR 95% CI U
Upper limit of 95% confidence interval of the allelic odds
ratio
Definition of entries in the "Stratification" and "Strata" columns
(Table 6) for stratification-based analyses:
TABLE-US-00002 Stratification Strata Definition no ALL All
individuals BMI_GE27 L/H Individuals with body mass index
lower/higher than 27 HTN Y/N Individuals with/without (Y/N) history
of hypertension SEX M/F Gender male/female AGE_LT60 Y/O Individuals
younger/ older than 60 years SMOKE Y/N Individuals that have/ do
not have history of smoking
Definition of entries in the "Status" column (Table 6):
TABLE-US-00003 Status Definition LT60MI_60TO75noMI MI cases younger
than 60 compared to controls between the ages of 60 to 75
LT60MI_GT75noMI MI cases younger than 60 compared to controls older
than 75 MI_GT75noMI MI cases compared to controls older than 75
MI_LT75noMI MI cases compared to controls younger than 75 MI_noASD
Cases with history of MI, controls with no history of
atherosclerotic disease MI_noMI Cases with history of MI, controls
with no history of MI MI_YOUNGOLD_noASD Cases with history of MI
under the age of 60 controls with no history of atherosclerotic
disease over the age of 60 MI_YOUNGOLD_noMI Cases with history of
MI under the age of 60 controls with no history of MI over the age
of 60 YoungMI_GT75noASD Cases with history of MI under the age of
60 controls with no history of atherosclerotic disease over the age
of 75
TABLE-US-00004 TABLE 7 (SNP association with Recurrent Myocardial
Infarction) Description of column headings for Table 7: Table 7
column heading Definition Gene Locus Link HUGO approved gene symbol
Marker Internal hCV identification number for the tested SNP Sample
Set Sample Set used in the analysis (CARE, Pre-CARE or WGS_S0012)
p-value Result of the asymptotic chi square test for allelic
association, dominant genotypic association, recessive genotypic
association, or the allelic, dominant, or recessive p-value of the
stratified analysis OR odds ratio 95% CI 95% confidence interval of
the given odds ratio Case_Freq Allele frequency of minor allele in
cases Control_Freq Allele frequency of minor allele in controls
Allele1 Nucleotide (allele) of the tested SNP for which statistics
are being reported Mode The mode of inheritance Strata Indicates if
the analysis of the dataset was based on a substratum such as
gender, age, BMI, Hypertension, Fasting Glucose levels, etc.
(strata are described below)
Definition of entries in the "Strata" column (Table 7) for
stratification-based analyses:
TABLE-US-00005 Stratum Definition BMI_TERTILE_1 Individuals in the
lowest tertile of body mass index BMI_TERTILE_2 Individuals in the
middle tertile of body mass index BMI_TERTILE_3 Individuals in the
highest tertile of body mass index PLACEBO Patients who were in
placebo arm of the CARE trail PRAVASTATIN Patients who were in
Pravastatin arm of the CARE trail MALE Only males FEMALE Only
females HYPERTEN_1 Individuals with history of Hypertension
HYPERTEN_0 Individuals without history of Hypertension
GLUCOSE_TERTILE_1 Individuals in a lowest tertile of Fasting
Glucose levels GLUCOSE_TERTILE_2 Individuals in a middle tertile of
Fasting Glucose levels GLUCOSE_TERTILE_3 Individuals in a highest
tertile of Fasting Glucose levels AGE_TERTILE_1 Individuals in a
lowest tertile of age (premature MI) AGE_TERTILE_2 Individuals in a
middle tertile of age AGE_TERTILE_3 Individuals in a highest
tertile age EVERSMOKED_0 Individuals who never smoked EVERSMOKED_1
Former smokers EVERSMOKED_2 Current smokers FMHX_CHD_1 Individuals
with family history of CHD FMHX_CHD_0 Individuals without family
history of CHD
DESCRIPTION OF THE FIGURE
FIG. 1 provides a diagrammatic representation of a computer-based
discovery system containing the SNP information of the present
invention in computer readable form.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides SNPs associated with myocardial
infarction (including recurrent myocardial infarction), nucleic
acid molecules containing SNPs, methods and reagents for the
detection of the SNPs disclosed herein, uses of these SNPs for the
development of detection reagents, and assays or kits that utilize
such reagents. The myocardial infarction-associated SNPs disclosed
herein are useful for diagnosing, screening for, and evaluating
predisposition to myocardial infarction and related pathologies in
humans. Furthermore, such SNPs and their encoded products are
useful targets for the development of therapeutic agents.
A large number of SNPs have been identified from re-sequencing DNA
from 39 individuals, and they are indicated as "Applera" SNP source
in Tables 1-2. Their allele frequencies observed in each of the
Caucasian and African-American ethnic groups are provided.
Additional SNPs included herein were previously identified during
shotgun sequencing and assembly of the human genome, and they are
indicated as "Celera" SNP source in Tables 1-2. Furthermore, the
information provided in Table 1-2, particularly the allele
frequency information obtained from 39 individuals and the
identification of the precise position of each SNP within each
gene/transcript, allows haplotypes (i.e., groups of SNPs that are
co-inherited) to be readily inferred. The present invention
encompasses SNP haplotypes, as well as individual SNPs.
Thus, the present invention provides individual SNPs associated
with myocardial infarction, as well as combinations of SNPs and
haplotypes in genetic regions associated with myocardial
infarction, polymorphic/variant transcript sequences (SEQ ID
NOS:1-828) and genomic sequences (SEQ ID NOS:17,553-18,016)
containing SNPs, encoded amino acid sequences (SEQ ID NOS:
829-1656), and both transcript-based SNP context sequences (SEQ ID
NOS: 1657-17,552) and genomic-based SNP context sequences (SEQ ID
NOS:18,017-73,085) (transcript sequences, protein sequences, and
transcript-based SNP context sequences are provided in Table 1 and
the Sequence Listing; genomic sequences and genomic-based SNP
context sequences are provided in Table 2 and the Sequence
Listing), methods of detecting these polymorphisms in a test
sample, methods of determining the risk of an individual of having
or developing myocardial infarction, methods of screening for
compounds useful for treating disorders associated with a variant
gene/protein such as myocardial infarction, compounds identified by
these screening methods, methods of using the disclosed SNPs to
select a treatment strategy, methods of treating a disorder
associated with a variant gene/protein (i.e., therapeutic methods),
and methods of using the SNPs of the present invention for human
identification.
The present invention provides novel SNPs associated with
myocardial infarction, as well as SNPs that were previously known
in the art, but were not previously known to be associated with
myocardial infarction. Accordingly, the present invention provides
novel compositions and methods based on the novel SNPs disclosed
herein, and also provides novel methods of using the known, but
previously unassociated, SNPs in methods relating to myocardial
infarction (e.g., for diagnosing myocardial infarction, etc.). In
Tables 1-2, known SNPs are identified based on the public database
in which they have been observed, which is indicated as one or more
of the following SNP types: "dbSNP"=SNP observed in dbSNP,
"HGBASE"=SNP observed in HGBASE, and "HGMD"=SNP observed in the
Human Gene Mutation Database (HGMD). Novel SNPs for which the SNP
source is only "Applera" and none other, i.e., those that have not
been observed in any public databases and which were also not
observed during shotgun sequencing and assembly of the Celera human
genome sequence (i.e., "Celera" SNP source), are indicated in
Tables 3-4.
Particular SNP alleles of the present invention can be associated
with either an increased risk of having or developing myocardial
infarction, or a decreased risk of having or developing myocardial
infarction. SNP alleles that are associated with a decreased risk
of having or developing myocardial infarction may be referred to as
"protective" alleles, and SNP alleles that are associated with an
increased risk of having or developing myocardial infarction may be
referred to as "susceptibility" alleles or "risk factors". Thus,
whereas certain SNPs (or their encoded products) can be assayed to
determine whether an individual possesses a SNP allele that is
indicative of an increased risk of having or developing myocardial
infarction (i.e., a susceptibility allele), other SNPs (or their
encoded products) can be assayed to determine whether an individual
possesses a SNP allele that is indicative of a decreased risk of
having or developing myocardial infarction (i.e., a protective
allele). Similarly, particular SNP alleles of the present invention
can be associated with either an increased or decreased likelihood
of responding to a particular treatment or therapeutic compound, or
an increased or decreased likelihood of experiencing toxic effects
from a particular treatment or therapeutic compound. The term
"altered" may be used herein to encompass either of these two
possibilities (e.g., an increased or a decreased
risk/likelihood).
Those skilled in the art will readily recognize that nucleic acid
molecules may be double-stranded molecules and that reference to a
particular site on one strand refers, as well, to the corresponding
site on a complementary strand. In defining a SNP position, SNP
allele, or nucleotide sequence, reference to an adenine, a thymine
(uridine), a cytosine, or a guanine at a particular site on one
strand of a nucleic acid molecule also defines the thymine
(uridine), adenine, guanine, or cytosine (respectively) at the
corresponding site on a complementary strand of the nucleic acid
molecule. Thus, reference may be made to either strand in order to
refer to a particular SNP position, SNP allele, or nucleotide
sequence. Probes and primers, may be designed to hybridize to
either strand and SNP genotyping methods disclosed herein may
generally target either strand. Throughout the specification, in
identifying a SNP position, reference is generally made to the
protein-encoding strand, only for the purpose of convenience.
References to variant peptides, polypeptides, or proteins of the
present invention include peptides, polypeptides, proteins, or
fragments thereof, that contain at least one amino acid residue
that differs from the corresponding amino acid sequence of the
art-known peptide/polypeptide/protein (the art-known protein may be
interchangeably referred to as the "wild-type", "reference", or
"normal" protein). Such variant peptides/polypeptides/proteins can
result from a codon change caused by a nonsynonymous nucleotide
substitution at a protein-coding SNP position (i.e., a missense
mutation) disclosed by the present invention. Variant
peptides/polypeptides/proteins of the present invention can also
result from a nonsense mutation, i.e. a SNP that creates a
premature stop codon, a SNP that generates a read-through mutation
by abolishing a stop codon, or due to any SNP disclosed by the
present invention that otherwise alters the structure,
function/activity, or expression of a protein, such as a SNP in a
regulatory region (e.g. a promoter or enhancer) or a SNP that leads
to alternative or defective splicing, such as a SNP in an intron or
a SNP at an exon/intron boundary. As used herein, the terms
"polypeptide", "peptide", and "protein" are used
interchangeably.
Isolated Nucleic Acid Molecules and SNP Detection Reagents &
Kits
Tables 1 and 2 provide a variety of information about each SNP of
the present invention that is associated with myocardial
infarction, including the transcript sequences (SEQ ID NOS:1-828),
genomic sequences (SEQ ID NOS:17,553-18,016), and protein sequences
(SEQ ID NOS:829-1656) of the encoded gene products (with the SNPs
indicated by IUB codes in the nucleic acid sequences). In addition,
Tables 1 and 2 include SNP context sequences, which generally
include 100 nucleotide upstream (5') plus 100 nucleotides
downstream (3') of each SNP position (SEQ ID NOS:1657-17,552
correspond to transcript-based SNP context sequences disclosed in
Table 1, and SEQ ID NOS:18,017-73,085 correspond to genomic-based
context sequences disclosed in Table 2), the alternative
nucleotides (alleles) at each SNP position, and additional
information about the variant where relevant, such as SNP type
(coding, missense, splice site, UTR, etc.), human populations in
which the SNP was observed, observed allele frequencies,
information about the encoded protein, etc.
Isolated Nucleic Acid Molecules
The present invention provides isolated nucleic acid molecules that
contain one or more SNPs disclosed Table 1 and/or Table 2.
Preferred isolated nucleic acid molecules contain one or more SNPs
identified in Table 3 and/or Table 4. Isolated nucleic acid
molecules containing one or more SNPs disclosed in at least one of
Tables 1-4 may be interchangeably referred to throughout the
present text as "SNP-containing nucleic acid molecules". Isolated
nucleic acid molecules may optionally encode a full-length variant
protein or fragment thereof. The isolated nucleic acid molecules of
the present invention also include probes and primers (which are
described in greater detail below in the section entitled "SNP
Detection Reagents"), which may be used for assaying the disclosed
SNPs, and isolated full-length genes, transcripts, cDNA molecules,
and fragments thereof, which may be used for such purposes as
expressing an encoded protein.
As used herein, an "isolated nucleic acid molecule" generally is
one that contains a SNP of the present invention or one that
hybridizes to such molecule such as a nucleic acid with a
complementary sequence, and is separated from most other nucleic
acids present in the natural source of the nucleic acid molecule.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule containing a SNP of the present invention, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or chemical precursors or
other chemicals when chemically synthesized. A nucleic acid
molecule can be fused to other coding or regulatory sequences and
still be considered "isolated". Nucleic acid molecules present in
non-human transgenic animals, which do not naturally occur in the
animal, are also considered "isolated". For example, recombinant
DNA molecules contained in a vector are considered "isolated".
Further examples of "isolated" DNA molecules include recombinant
DNA molecules maintained in heterologous host cells, and purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
isolated SNP-containing DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
Generally, an isolated SNP-containing nucleic acid molecule
comprises one or more SNP positions disclosed by the present
invention with flanking nucleotide sequences on either side of the
SNP positions. A flanking sequence can include nucleotide residues
that are naturally associated with the SNP site and/or heterologous
nucleotide sequences. Preferably the flanking sequence is up to
about 500, 300, 100, 60, 50, 30, 25, 20, 15, 10, 8, or 4
nucleotides (or any other length in-between) on either side of a
SNP position, or as long as the full-length gene or entire
protein-coding sequence (or any portion thereof such as an exon),
especially if the SNP-containing nucleic acid molecule is to be
used to produce a protein or protein fragment.
For full-length genes and entire protein-coding sequences, a SNP
flanking sequence can be, for example, up to about 5 KB, 4 KB, 3
KB, 2 KB, 1 KB on either side of the SNP. Furthermore, in such
instances, the isolated nucleic acid molecule comprises exonic
sequences (including protein-coding and/or non-coding exonic
sequences), but may also include intronic sequences. Thus, any
protein coding sequence may be either contiguous or separated by
introns. The important point is that the nucleic acid is isolated
from remote and unimportant flanking sequences and is of
appropriate length such that it can be subjected to the specific
manipulations or uses described herein such as recombinant protein
expression, preparation of probes and primers for assaying the SNP
position, and other uses specific to the SNP-containing nucleic
acid sequences.
An isolated SNP-containing nucleic acid molecule can comprise, for
example, a full-length gene or transcript, such as a gene isolated
from genomic DNA (e.g., by cloning or PCR amplification), a cDNA
molecule, or an mRNA transcript molecule. Polymorphic transcript
sequences are provided in Table 1 and in the Sequence Listing (SEQ
ID NOS: 1-828), and polymorphic genomic sequences are provided in
Table 2 and in the Sequence Listing (SEQ ID NOS:17,553-18,016).
Furthermore, fragments of such full-length genes and transcripts
that contain one or more SNPs disclosed herein are also encompassed
by the present invention, and such fragments may be used, for
example, to express any part of a protein, such as a particular
functional domain or an antigenic epitope.
Thus, the present invention also encompasses fragments of the
nucleic acid sequences provided in Tables 1-2 (transcript sequences
are provided in Table 1 as SEQ ID NOS:1-828, genomic sequences are
provided in Table 2 as SEQ ID NOS:17,553-18,016, transcript-based
SNP context sequences are provided in Table 1 as SEQ ID
NO:1657-17,552, and genomic-based SNP context sequences are
provided in Table 2 as SEQ ID NO:18,017-73,085) and their
complements. A fragment typically comprises a contiguous nucleotide
sequence at least about 8 or more nucleotides, more preferably at
least about 12 or more nucleotides, and even more preferably at
least about 16 or more nucleotides. Further, a fragment could
comprise at least about 18, 20, 22, 25, 30, 40, 50, 60, 100, 250 or
500 (or any other number in-between) nucleotides in length. The
length of the fragment will be based on its intended use. For
example, the fragment can encode epitope-bearing regions of a
variant peptide or regions of a variant peptide that differ from
the normal/wild-type protein, or can be useful as a polynucleotide
probe or primer. Such fragments can be isolated using the
nucleotide sequences provided in Table 1 and/or Table 2 for the
synthesis of a polynucleotide probe. A labeled probe can then be
used, for example, to screen a cDNA library, genomic DNA library,
or mRNA to isolate nucleic acid corresponding to the coding region.
Further, primers can be used in amplification reactions, such as
for purposes of assaying one or more SNPs sites or for cloning
specific regions of a gene.
An isolated nucleic acid molecule of the present invention further
encompasses a SNP-containing polynucleotide that is the product of
any one of a variety of nucleic acid amplification methods, which
are used to increase the copy numbers of a polynucleotide of
interest in a nucleic acid sample. Such amplification methods are
well known in the art, and they include but are not limited to,
polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195; and
4,683,202; PCR Technology: Principles and Applications for DNA
Amplification, ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992),
ligase chain reaction (LCR) (Wu and Wallace, Genomics 4:560, 1989;
Landegren et al., Science 241:1077, 1988), strand displacement
amplification (SDA) (U.S. Pat. Nos. 5,270,184; and 5,422,252),
transcription-mediated amplification (TMA) (U.S. Pat. No.
5,399,491), linked linear amplification (LLA) (U.S. Pat. No.
6,027,923), and the like, and isothermal amplification methods such
as nucleic acid sequence based amplification (NASBA), and
self-sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87: 1874, 1990). Based on such methodologies, a
person skilled in the art can readily design primers in any
suitable regions 5' and 3' to a SNP disclosed herein. Such primers
may be used to amplify DNA of any length so long that it contains
the SNP of interest in its sequence.
As used herein, an "amplified polynucleotide" of the invention is a
SNP-containing nucleic acid molecule whose amount has been
increased at least two fold by any nucleic acid amplification
method performed in vitro as compared to its starting amount in a
test sample. In other preferred embodiments, an amplified
polynucleotide is the result of at least ten fold, fifty fold, one
hundred fold, one thousand fold, or even ten thousand fold increase
as compared to its starting amount in a test sample. In a typical
PCR amplification, a polynucleotide of interest is often amplified
at least fifty thousand fold in amount over the unamplified genomic
DNA, but the precise amount of amplification needed for an assay
depends on the sensitivity of the subsequent detection method
used.
Generally, an amplified polynucleotide is at least about 16
nucleotides in length. More typically, an amplified polynucleotide
is at least about 20 nucleotides in length. In a preferred
embodiment of the invention, an amplified polynucleotide is at
least about 30 nucleotides in length. In a more preferred
embodiment of the invention, an amplified polynucleotide is at
least about 32, 40, 45, 50, or 60 nucleotides in length. In yet
another preferred embodiment of the invention, an amplified
polynucleotide is at least about 100, 200, or 300 nucleotides in
length. While the total length of an amplified polynucleotide of
the invention can be as long as an exon, an intron or the entire
gene where the SNP of interest resides, an amplified product is
typically no greater than about 1,000 nucleotides in length
(although certain amplification methods may generate amplified
products greater than 1000 nucleotides in length). More preferably,
an amplified polynucleotide is not greater than about 600
nucleotides in length. It is understood that irrespective of the
length of an amplified polynucleotide, a SNP of interest may be
located anywhere along its sequence.
In a specific embodiment of the invention, the amplified product is
at least about 201 nucleotides in length, comprises one of the
transcript-based context sequences or the genomic-based context
sequences shown in Tables 1-2. Such a product may have additional
sequences on its 5' end or 3' end or both. In another embodiment,
the amplified product is about 101 nucleotides in length, and it
contains a SNP disclosed herein. Preferably, the SNP is located at
the middle of the amplified product (e.g., at position 101 in an
amplified product that is 201 nucleotides in length, or at position
51 in an amplified product that is 101 nucleotides in length), or
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 nucleotides
from the middle of the amplified product (however, as indicated
above, the SNP of interest may be located anywhere along the length
of the amplified product).
The present invention provides isolated nucleic acid molecules that
comprise, consist of, or consist essentially of one or more
polynucleotide sequences that contain one or more SNPs disclosed
herein, complements thereof, and SNP-containing fragments
thereof.
Accordingly, the present invention provides nucleic acid molecules
that consist of any of the nucleotide sequences shown in Table 1
and/or Table 2 (transcript sequences are provided in Table 1 as SEQ
ID NOS:1-828, genomic sequences are provided in Table 2 as SEQ ID
NOS:17,553-18,016, transcript-based SNP context sequences are
provided in Table 1 as SEQ ID NO:1657-17,552, and genomic-based SNP
context sequences are provided in Table 2 as SEQ ID
NO:18,017-73,085), or any nucleic acid molecule that encodes any of
the variant proteins provided in Table 1 (SEQ ID NOS:829-1656). A
nucleic acid molecule consists of a nucleotide sequence when the
nucleotide sequence is the complete nucleotide sequence of the
nucleic acid molecule.
The present invention further provides nucleic acid molecules that
consist essentially of any of the nucleotide sequences shown in
Table 1 and/or Table 2 (transcript sequences are provided in Table
1 as SEQ ID NOS:1-828, genomic sequences are provided in Table 2 as
SEQ ID NOS:17,553-18,016, transcript-based SNP context sequences
are provided in Table 1 as SEQ ID NO:1657-17,552, and genomic-based
SNP context sequences are provided in Table 2 as SEQ ID
NO:18,017-73,085), or any nucleic acid molecule that encodes any of
the variant proteins provided in Table 1 (SEQ ID NOS:829-1656). A
nucleic acid molecule consists essentially of a nucleotide sequence
when such a nucleotide sequence is present with only a few
additional nucleotide residues in the final nucleic acid
molecule.
The present invention further provides nucleic acid molecules that
comprise any of the nucleotide sequences shown in Table 1 and/or
Table 2 or a SNP-containing fragment thereof (transcript sequences
are provided in Table 1 as SEQ ID NOS:1-828, genomic sequences are
provided in Table 2 as SEQ ID NOS:17,553-18,016, transcript-based
SNP context sequences are provided in Table 1 as SEQ ID
NO:1657-17,552, and genomic-based SNP context sequences are
provided in Table 2 as SEQ ID NO:18,017-73,085), or any nucleic
acid molecule that encodes any of the variant proteins provided in
Table 1 (SEQ ID NOS:829-1656). A nucleic acid molecule comprises a
nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleotide residues, such as
residues that are naturally associated with it or heterologous
nucleotide sequences. Such a nucleic acid molecule can have one to
a few additional nucleotides or can comprise many more additional
nucleotides. A brief description of how various types of these
nucleic acid molecules can be readily made and isolated is provided
below, and such techniques are well known to those of ordinary
skill in the art (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY).
The isolated nucleic acid molecules can encode mature proteins plus
additional amino or carboxyl-terminal amino acids or both, or amino
acids interior to the mature peptide (when the mature form has more
than one peptide chain, for instance). Such sequences may play a
role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life, or facilitate manipulation of a protein for assay or
production. As generally is the case in situ, the additional amino
acids may be processed away from the mature protein by cellular
enzymes.
Thus, the isolated nucleic acid molecules include, but are not
limited to, nucleic acid molecules having a sequence encoding a
peptide alone, a sequence encoding a mature peptide and additional
coding sequences such as a leader or secretory sequence (e.g., a
pre-pro or pro-protein sequence), a sequence encoding a mature
peptide with or without additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but untranslated sequences
that play a role in, for example, transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding,
and/or stability of mRNA. In addition, the nucleic acid molecules
may be fused to heterologous marker sequences encoding, for
example, a peptide that facilitates purification.
Isolated nucleic acid molecules can be in the form of RNA, such as
mRNA, or in the form DNA, including cDNA and genomic DNA, which may
be obtained, for example, by molecular cloning or produced by
chemical synthetic techniques or by a combination thereof (Sambrook
and Russell, 2000, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press, NY). Furthermore, isolated nucleic acid
molecules, particularly SNP detection reagents such as probes and
primers, can also be partially or completely in the form of one or
more types of nucleic acid analogs, such as peptide nucleic acid
(PNA) (U.S. Pat. Nos. 5,539,082; 5,527,675; 5,623,049; 5,714,331).
The nucleic acid, especially DNA, can be double-stranded or
single-stranded. Single-stranded nucleic acid can be the coding
strand (sense strand) or the complementary non-coding strand
(anti-sense strand). DNA, RNA, or PNA segments can be assembled,
for example, from fragments of the human genome (in the case of DNA
or RNA) or single nucleotides, short oligonucleotide linkers, or
from a series of oligonucleotides, to provide a synthetic nucleic
acid molecule. Nucleic acid molecules can be readily synthesized
using the sequences provided herein as a reference; oligonucleotide
and PNA oligomer synthesis techniques are well known in the art
(see, e.g., Corey, "Peptide nucleic acids: expanding the scope of
nucleic acid recognition", Trends Biotechnol. 1997 June;
15(6):224-9, and Hyrup et al., "Peptide nucleic acids (PNA):
synthesis, properties and potential applications", Bioorg Med Chem.
1996 January; 4(1):5-23). Furthermore, large-scale automated
oligonucleotide/PNA synthesis (including synthesis on an array or
bead surface or other solid support) can readily be accomplished
using commercially available nucleic acid synthesizers, such as the
Applied Biosystems (Foster City, Calif.) 3900 High-Throughput DNA
Synthesizer or Expedite 8909 Nucleic Acid Synthesis System, and the
sequence information provided herein.
The present invention encompasses nucleic acid analogs that contain
modified, synthetic, or non-naturally occurring nucleotides or
structural elements or other alternative/modified nucleic acid
chemistries known in the art. Such nucleic acid analogs are useful,
for example, as detection reagents (e.g., primers/probes) for
detecting one or more SNPs identified in Table 1 and/or Table 2.
Furthermore, kits/systems (such as beads, arrays, etc.) that
include these analogs are also encompassed by the present
invention. For example, PNA oligomers that are based on the
polymorphic sequences of the present invention are specifically
contemplated. PNA oligomers are analogs of DNA in which the
phosphate backbone is replaced with a peptide-like backbone
(Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,
4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996), Kumar et al., Organic Letters
3(9): 1269-1272 (2001), WO96/04000). PNA hybridizes to
complementary RNA or DNA with higher affinity and specificity than
conventional oligonucleotides and oligonucleotide analogs. The
properties of PNA enable novel molecular biology and biochemistry
applications unachievable with traditional oligonucleotides and
peptides.
Additional examples of nucleic acid modifications that improve the
binding properties and/or stability of a nucleic acid include the
use of base analogs such as inosine, intercalators (U.S. Pat. No.
4,835,263) and the minor groove binders (U.S. Pat. No. 5,801,115).
Thus, references herein to nucleic acid molecules, SNP-containing
nucleic acid molecules, SNP detection reagents (e.g., probes and
primers), oligonucleotides/polynucleotides include PNA oligomers
and other nucleic acid analogs. Other examples of nucleic acid
analogs and alternative/modified nucleic acid chemistries known in
the art are described in Current Protocols in Nucleic Acid
Chemistry, John Wiley & Sons, N.Y. (2002).
The present invention further provides nucleic acid molecules that
encode fragments of the variant polypeptides disclosed herein as
well as nucleic acid molecules that encode obvious variants of such
variant polypeptides. Such nucleic acid molecules may be naturally
occurring, such as paralogs (different locus) and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Non-naturally occurring variants
may be made by mutagenesis techniques, including those applied to
nucleic acid molecules, cells, or organisms. Accordingly, the
variants can contain nucleotide substitutions, deletions,
inversions and insertions (in addition to the SNPs disclosed in
Tables 1-2). Variation can occur in either or both the coding and
non-coding regions. The variations can produce conservative and/or
non-conservative amino acid substitutions.
Further variants of the nucleic acid molecules disclosed in Tables
1-2, such as naturally occurring allelic variants (as well as
orthologs and paralogs) and synthetic variants produced by
mutagenesis techniques, can be identified and/or produced using
methods well known in the art. Such further variants can comprise a
nucleotide sequence that shares at least 70-80%, 80-85%, 85-90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
with a nucleic acid sequence disclosed in Table 1 and/or Table 2
(or a fragment thereof) and that includes a novel SNP allele
disclosed in Table 1 and/or Table 2. Further, variants can comprise
a nucleotide sequence that encodes a polypeptide that shares at
least 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity with a polypeptide sequence disclosed
in Table 1 (or a fragment thereof) and that includes a novel SNP
allele disclosed in Table 1 and/or Table 2. Thus, the present
invention specifically contemplates isolated nucleic acid molecule
that have a certain degree of sequence variation compared with the
sequences shown in Tables 1-2, but that contain a novel SNP allele
disclosed herein. In other words, as long as an isolated nucleic
acid molecule contains a novel SNP allele disclosed herein, other
portions of the nucleic acid molecule that flank the novel SNP
allele can vary to some degree from the specific transcript,
genomic, and context sequences shown in Tables 1-2, and can encode
a polypeptide that varies to some degree from the specific
polypeptide sequences shown in Table 1.
To determine the percent identity of two amino acid sequences or
two nucleotide sequences of two molecules that share sequence
homology, the sequences are aligned for optimal comparison purposes
(e.g., gaps can be introduced in one or both of a first and a
second amino acid or nucleic acid sequence for optimal alignment
and non-homologous sequences can be disregarded for comparison
purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%,
70%, 80%, or 90% or more of the length of a reference sequence is
aligned for comparison purposes. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein, amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
The comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. (Computational Molecular Biology, Lesk, A. M., ed.,
Oxford University Press, New York, 1988; Biocomputing: Informatics
and Genome Projects, Smith, D. W., ed., Academic Press, New York,
1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,
and Griffin, H. G., eds., Humana Press, N. J., 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991). In a preferred embodiment,
the percent identity between two amino acid sequences is determined
using the Needleman and Wunsch algorithm (J. Mol. Biol.
(48):444-453 (1970)) which has been incorporated into the GAP
program in the GCG software package, using either a Blossom 62
matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,
6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
In yet another preferred embodiment, the percent identity between
two nucleotide sequences is determined using the GAP program in the
GCG software package (Devereux, J., et al., Nucleic Acids Res.
12(1):387 (1984)), using a NWSgapdna.CMP matrix and a gap weight of
40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
In another embodiment, the percent identity between two amino acid
or nucleotide sequences is determined using the algorithm of E.
Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12, and a gap penalty
of 4.
The nucleotide and amino acid sequences of the present invention
can further be used as a "query sequence" to perform a search
against sequence databases to, for example, identify other family
members or related sequences. Such searches can be performed using
the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.
(J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. In addition to BLAST, examples of
other search and sequence comparison programs used in the art
include, but are not limited to, FASTA (Pearson, Methods Mol. Biol.
25, 365-389 (1994)) and KERR (Dufresne et al., Nat Biotechnol 2002
December; 20(12):1269-71). For further information regarding
bioinformatics techniques, see Current Protocols in Bioinformatics,
John Wiley & Sons, Inc., N.Y.
The present invention further provides non-coding fragments of the
nucleic acid molecules disclosed in Table 1 and/or Table 2.
Preferred non-coding fragments include, but are not limited to,
promoter sequences, enhancer sequences, intronic sequences, 5'
untranslated regions (UTRs), 3' untranslated regions, gene
modulating sequences and gene termination sequences. Such fragments
are useful, for example, in controlling heterologous gene
expression and in developing screens to identify gene-modulating
agents.
SNP Detection Reagents
In a specific aspect of the present invention, the SNPs disclosed
in Table 1 and/or Table 2, and their associated transcript
sequences (provided in Table 1 as SEQ ID NOS:1-828), genomic
sequences (provided in Table 2 as SEQ ID NOS:17,553-18,016), and
context sequences (transcript-based context sequences are provided
in Table 1 as SEQ ID NOS:1657-17,552; genomic-based context
sequences are provided in Table 2 as SEQ ID NOS:18,017-73,085), can
be used for the design of SNP detection reagents. As used herein, a
"SNP detection reagent" is a reagent that specifically detects a
specific target SNP position disclosed herein, and that is
preferably specific for a particular nucleotide (allele) of the
target SNP position (i.e., the detection reagent preferably can
differentiate between different alternative nucleotides at a target
SNP position, thereby allowing the identity of the nucleotide
present at the target SNP position to be determined). Typically,
such detection reagent hybridizes to a target SNP-containing
nucleic acid molecule by complementary base-pairing in a sequence
specific manner, and discriminates the target variant sequence from
other nucleic acid sequences such as an art-known form in a test
sample. An example of a detection reagent is a probe that
hybridizes to a target nucleic acid containing one or more of the
SNPs provided in Table 1 and/or Table 2. In a preferred embodiment,
such a probe can differentiate between nucleic acids having a
particular nucleotide (allele) at a target SNP position from other
nucleic acids that have a different nucleotide at the same target
SNP position. In addition, a detection reagent may hybridize to a
specific region 5' and/or 3' to a SNP position, particularly a
region corresponding to the context sequences provided in Table 1
and/or Table 2 (transcript-based context sequences are provided in
Table 1 as SEQ ID NOS:1657-17,552; genomic-based context sequences
are provided in Table 2 as SEQ ID NOS:18,017-73,085). Another
example of a detection reagent is a primer which acts as an
initiation point of nucleotide extension along a complementary
strand of a target polynucleotide. The SNP sequence information
provided herein is also useful for designing primers, e.g.
allele-specific primers, to amplify (e.g., using PCR) any SNP of
the present invention.
In one preferred embodiment of the invention, a SNP detection
reagent is an isolated or synthetic DNA or RNA polynucleotide probe
or primer or PNA oligomer, or a combination of DNA, RNA and/or PNA,
that hybridizes to a segment of a target nucleic acid molecule
containing a SNP identified in Table 1 and/or Table 2. A detection
reagent in the form of a polynucleotide may optionally contain
modified base analogs, intercalators or minor groove binders.
Multiple detection reagents such as probes may be, for example,
affixed to a solid support (e.g., arrays or beads) or supplied in
solution (e.g., probe/primer sets for enzymatic reactions such as
PCR, RT-PCR, TaqMan assays, or primer-extension reactions) to form
a SNP detection kit.
A probe or primer typically is a substantially purified
oligonucleotide or PNA oligomer. Such oligonucleotide typically
comprises a region of complementary nucleotide sequence that
hybridizes under stringent conditions to at least about 8, 10, 12,
16, 18, 20, 22, 25, 30, 40, 50, 60, 100 (or any other number
in-between) or more consecutive nucleotides in a target nucleic
acid molecule. Depending on the particular assay, the consecutive
nucleotides can either include the target SNP position, or be a
specific region in close enough proximity 5' and/or 3' to the SNP
position to carry out the desired assay.
Other preferred primer and probe sequences can readily be
determined using the transcript sequences (SEQ ID NOS:1-828),
genomic sequences (SEQ ID NOS:17,553-18,016), and SNP context
sequences (transcript-based context sequences are provided in Table
1 as SEQ ID NOS:1657-17,552; genomic-based context sequences are
provided in Table 2 as SEQ ID NOS:18,017-73,085) disclosed in the
Sequence Listing and in Tables 1-2. It will be apparent to one of
skill in the art that such primers and probes are directly useful
as reagents for genotyping the SNPs of the present invention, and
can be incorporated into any kit/system format.
In order to produce a probe or primer specific for a target
SNP-containing sequence, the gene/transcript and/or context
sequence surrounding the SNP of interest is typically examined
using a computer algorithm which starts at the 5' or at the 3' end
of the nucleotide sequence. Typical algorithms will then identify
oligomers of defined length that are unique to the gene/SNP context
sequence, have a GC content within a range suitable for
hybridization, lack predicted secondary structure that may
interfere with hybridization, and/or possess other desired
characteristics or that lack other undesired characteristics.
A primer or probe of the present invention is typically at least
about 8 nucleotides in length. In one embodiment of the invention,
a primer or a probe is at least about 10 nucleotides in length. In
a preferred embodiment, a primer or a probe is at least about 12
nucleotides in length. In a more preferred embodiment, a primer or
probe is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25
nucleotides in length. While the maximal length of a probe can be
as long as the target sequence to be detected, depending on the
type of assay in which it is employed, it is typically less than
about 50, 60, 65, or 70 nucleotides in length. In the case of a
primer, it is typically less than about 30 nucleotides in length.
In a specific preferred embodiment of the invention, a primer or a
probe is within the length of about 18 and about 28 nucleotides.
However, in other embodiments, such as nucleic acid arrays and
other embodiments in which probes are affixed to a substrate, the
probes can be longer, such as on the order of 30-70, 75, 80, 90,
100, or more nucleotides in length (see the section below entitled
"SNP Detection Kits and Systems").
For analyzing SNPs, it may be appropriate to use oligonucleotides
specific for alternative SNP alleles. Such oligonucleotides which
detect single nucleotide variations in target sequences may be
referred to by such terms as "allele-specific oligonucleotides",
"allele-specific probes", or "allele-specific primers". The design
and use of allele-specific probes for analyzing polymorphisms is
described in, e.g., Mutation Detection A Practical Approach, ed.
Cotton et al. Oxford University Press, 1998; Saiki et al., Nature
324, 163-166 (1986); Dattagupta, EP235,726; and Saiki, WO
89/11548.
While the design of each allele-specific primer or probe depends on
variables such as the precise composition of the nucleotide
sequences flanking a SNP position in a target nucleic acid
molecule, and the length of the primer or probe, another factor in
the use of primers and probes is the stringency of the condition
under which the hybridization between the probe or primer and the
target sequence is performed. Higher stringency conditions utilize
buffers with lower ionic strength and/or a higher reaction
temperature, and tend to require a more perfect match between
probe/primer and a target sequence in order to form a stable
duplex. If the stringency is too high, however, hybridization may
not occur at all. In contrast, lower stringency conditions utilize
buffers with higher ionic strength and/or a lower reaction
temperature, and permit the formation of stable duplexes with more
mismatched bases between a probe/primer and a target sequence. By
way of example and not limitation, exemplary conditions for high
stringency hybridization conditions using an allele-specific probe
are as follows: Prehybridization with a solution containing
5.times. standard saline phosphate EDTA (SSPE), 0.5% NaDodSO.sub.4
(SDS) at 55.degree. C., and incubating probe with target nucleic
acid molecules in the same solution at the same temperature,
followed by washing with a solution containing 2.times.SSPE, and
0.1% SDS at 55.degree. C. or room temperature.
Moderate stringency hybridization conditions may be used for
allele-specific primer extension reactions with a solution
containing, e.g., about 50 mM KCl at about 46.degree. C.
Alternatively, the reaction may be carried out at an elevated
temperature such as 60.degree. C. In another embodiment, a
moderately stringent hybridization condition suitable for
oligonucleotide ligation assay (OLA) reactions wherein two probes
are ligated if they are completely complementary to the target
sequence may utilize a solution of about 100 mM KCl at a
temperature of 46.degree. C.
In a hybridization-based assay, allele-specific probes can be
designed that hybridize to a segment of target DNA from one
individual but do not hybridize to the corresponding segment from
another individual due to the presence of different polymorphic
forms (e.g., alternative SNP alleles/nucleotides) in the respective
DNA segments from the two individuals. Hybridization conditions
should be sufficiently stringent that there is a significant
detectable difference in hybridization intensity between alleles,
and preferably an essentially binary response, whereby a probe
hybridizes to only one of the alleles or significantly more
strongly to one allele. While a probe may be designed to hybridize
to a target sequence that contains a SNP site such that the SNP
site aligns anywhere along the sequence of the probe, the probe is
preferably designed to hybridize to a segment of the target
sequence such that the SNP site aligns with a central position of
the probe (e.g., a position within the probe that is at least three
nucleotides from either end of the probe). This design of probe
generally achieves good discrimination in hybridization between
different allelic forms.
In another embodiment, a probe or primer may be designed to
hybridize to a segment of target DNA such that the SNP aligns with
either the 5' most end or the 3' most end of the probe or primer.
In a specific preferred embodiment which is particularly suitable
for use in a oligonucleotide ligation assay (U.S. Pat. No.
4,988,617), the most nucleotide of the probe aligns with the SNP
position in the target sequence.
Oligonucleotide probes and primers may be prepared by methods well
known in the art. Chemical synthetic methods include, but are
limited to, the phosphotriester method described by Narang et al.,
1979, Methods in Enzymology 68:90; the phosphodiester method
described by Brown et al., 1979, Methods in Enzymology 68:109, the
diethylphosphoamidate method described by Beaucage et al., 1981,
Tetrahedron Letters 22:1859; and the solid support method described
in U.S. Pat. No. 4,458,066.
Allele-specific probes are often used in pairs (or, less commonly,
in sets of 3 or 4, such as if a SNP position is known to have 3 or
4 alleles, respectively, or to assay both strands of a nucleic acid
molecule for a target SNP allele), and such pairs may be identical
except for a one nucleotide mismatch that represents the allelic
variants at the SNP position. Commonly, one member of a pair
perfectly matches a reference form of a target sequence that has a
more common SNP allele (i.e., the allele that is more frequent in
the target population) and the other member of the pair perfectly
matches a form of the target sequence that has a less common SNP
allele (i.e., the allele that is rarer in the target population).
In the case of an array, multiple pairs of probes can be
immobilized on the same support for simultaneous analysis of
multiple different polymorphisms.
In one type of PCR-based assay, an allele-specific primer
hybridizes to a region on a target nucleic acid molecule that
overlaps a SNP position and only primes amplification of an allelic
form to which the primer exhibits perfect complementarity (Gibbs,
1989, Nucleic Acid Res. 17 2427-2448). Typically, the primer's
3'-most nucleotide is aligned with and complementary to the SNP
position of the target nucleic acid molecule. This primer is used
in conjunction with a second primer that hybridizes at a distal
site. Amplification proceeds from the two primers, producing a
detectable product that indicates which allelic form is present in
the test sample. A control is usually performed with a second pair
of primers, one of which shows a single base mismatch at the
polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch prevents
amplification or substantially reduces amplification efficiency, so
that either no detectable product is formed or it is formed in
lower amounts or at a slower pace. The method generally works most
effectively when the mismatch is at the 3'-most position of the
oligonucleotide (i.e., the 3'-most position of the oligonucleotide
aligns with the target SNP position) because this position is most
destabilizing to elongation from the primer (see, e.g., WO
93/22456). This PCR-based assay can be utilized as part of the
TaqMan assay, described below.
In a specific embodiment of the invention, a primer of the
invention contains a sequence substantially complementary to a
segment of a target SNP-containing nucleic acid molecule except
that the primer has a mismatched nucleotide in one of the three
nucleotide positions at the 3'-most end of the primer, such that
the mismatched nucleotide does not base pair with a particular
allele at the SNP site. In a preferred embodiment, the mismatched
nucleotide in the primer is the second from the last nucleotide at
the 3'-most position of the primer. In a more preferred embodiment,
the mismatched nucleotide in the primer is the last nucleotide at
the 3'-most position of the primer.
In another embodiment of the invention, a SNP detection reagent of
the invention is labeled with a fluorogenic reporter dye that emits
a detectable signal. While the preferred reporter dye is a
fluorescent dye, any reporter dye that can be attached to a
detection reagent such as an oligonucleotide probe or primer is
suitable for use in the invention. Such dyes include, but are not
limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5,
Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet,
Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and
Texas Red.
In yet another embodiment of the invention, the detection reagent
may be further labeled with a quencher dye such as Tamra,
especially when the reagent is used as a self-quenching probe such
as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular
Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other
stemless or linear beacon probe (Livak et al., 1995, PCR Method
Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14:
303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521;
U.S. Pat. Nos. 5,866,336 and 6,117,635).
The detection reagents of the invention may also contain other
labels, including but not limited to, biotin for streptavidin
binding, hapten for antibody binding, and oligonucleotide for
binding to another complementary oligonucleotide such as pairs of
zipcodes.
The present invention also contemplates reagents that do not
contain (or that are complementary to) a SNP nucleotide identified
herein but that are used to assay one or more SNPs disclosed
herein. For example, primers that flank, but do not hybridize
directly to a target SNP position provided herein are useful in
primer extension reactions in which the primers hybridize to a
region adjacent to the target SNP position (i.e., within one or
more nucleotides from the target SNP site). During the primer
extension reaction, a primer is typically not able to extend past a
target SNP site if a particular nucleotide (allele) is present at
that target SNP site, and the primer extension product can readily
be detected in order to determine which SNP allele is present at
the target SNP site. For example, particular ddNTPs are typically
used in the primer extension reaction to terminate primer extension
once a ddNTP is incorporated into the extension product (a primer
extension product which includes a ddNTP at the 3'-most end of the
primer extension product, and in which the ddNTP corresponds to a
SNP disclosed herein, is a composition that is encompassed by the
present invention). Thus, reagents that bind to a nucleic acid
molecule in a region adjacent to a SNP site, even though the bound
sequences do not necessarily include the SNP site itself, are also
encompassed by the present invention.
SNP Detection Kits and Systems
A person skilled in the art will recognize that, based on the SNP
and associated sequence information disclosed herein, detection
reagents can be developed and used to assay any SNP of the present
invention individually or in combination, and such detection
reagents can be readily incorporated into one of the established
kit or system formats which are well known in the art. The terms
"kits" and "systems", as used herein in the context of SNP
detection reagents, are intended to refer to such things as
combinations of multiple SNP detection reagents, or one or more SNP
detection reagents in combination with one or more other types of
elements or components (e.g., other types of biochemical reagents,
containers, packages such as packaging intended for commercial
sale, substrates to which SNP detection reagents are attached,
electronic hardware components, etc.). Accordingly, the present
invention further provides SNP detection kits and systems,
including but not limited to, packaged probe and primer sets (e.g.,
TaqMan probe/primer sets), arrays/microarrays of nucleic acid
molecules, and beads that contain one or more probes, primers, or
other detection reagents for detecting one or more SNPs of the
present invention. The kits/systems can optionally include various
electronic hardware components; for example, arrays ("DNA chips")
and microfluidic systems ("lab-on-a-chip" systems) provided by
various manufacturers typically comprise hardware components. Other
kits/systems (e.g., probe/primer sets) may not include electronic
hardware components, but may be comprised of, for example, one or
more SNP detection reagents (along with, optionally, other
biochemical reagents) packaged in one or more containers.
In some embodiments, a SNP detection kit typically contains one or
more detection reagents and other components (e.g., a buffer,
enzymes such as DNA polymerases or ligases, chain extension
nucleotides such as deoxynucleotide triphosphates, and in the case
of Sanger-type DNA sequencing reactions, chain terminating
nucleotides, positive control sequences, negative control
sequences, and the like) necessary to carry out an assay or
reaction, such as amplification and/or detection of a
SNP-containing nucleic acid molecule. A kit may further contain
means for determining the amount of a target nucleic acid, and
means for comparing the amount with a standard, and can comprise
instructions for using the kit to detect the SNP-containing nucleic
acid molecule of interest. In one embodiment of the present
invention, kits are provided which contain the necessary reagents
to carry out one or more assays to detect one or more SNPs
disclosed herein. In a preferred embodiment of the present
invention, SNP detection kits/systems are in the form of nucleic
acid arrays, or compartmentalized kits, including
microfluidic/lab-on-a-chip systems.
SNP detection kits/systems may contain, for example, one or more
probes, or pairs of probes, that hybridize to a nucleic acid
molecule at or near each target SNP position. Multiple pairs of
allele-specific probes may be included in the kit/system to
simultaneously assay large numbers of SNPs, at least one of which
is a SNP of the present invention. In some kits/systems, the
allele-specific probes are immobilized to a substrate such as an
array or bead. For example, the same substrate can comprise
allele-specific probes for detecting at least 1; 10; 100; 1000;
10,000; 100,000 (or any other number in-between) or substantially
all of the SNPs shown in Table 1 and/or Table 2.
The terms "arrays", "microarrays", and "DNA chips" are used herein
interchangeably to refer to an array of distinct polynucleotides
affixed to a substrate, such as glass, plastic, paper, nylon or
other type of membrane, filter, chip, or any other suitable solid
support. The polynucleotides can be synthesized directly on the
substrate, or synthesized separate from the substrate and then
affixed to the substrate. In one embodiment, the microarray is
prepared and used according to the methods described in U.S. Pat.
No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et
al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680)
and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619), all of which are incorporated herein in their
entirety by reference. In other embodiments, such arrays are
produced by the methods described by Brown et al., U.S. Pat. No.
5,807,522.
Nucleic acid arrays are reviewed in the following references:
Zammatteo et al., "New chips for molecular biology and
diagnostics", Biotechnol Annu Rev. 2002; 8:85-101; Sosnowski et
al., "Active microelectronic array system for DNA hybridization,
genotyping and pharmacogenomic applications", Psychiatr Genet. 2002
December; 12(4):181-92; Heller, "DNA microarray technology:
devices, systems, and applications", Annu Rev Biomed Eng. 2002;
4:129-53. Epub 2002 Mar. 22; Kolchinsky et al., "Analysis of SNPs
and other genomic variations using gel-based chips", Hum Mutat.
2002 April; 19(4):343-60; and McGall et al., "High-density genechip
oligonucleotide probe arrays", Adv Biochem Eng Biotechnol. 2002;
77:21-42.
Any number of probes, such as allele-specific probes, may be
implemented in an array, and each probe or pair of probes can
hybridize to a different SNP position. In the case of
polynucleotide probes, they can be synthesized at designated areas
(or synthesized separately and then affixed to designated areas) on
a substrate using a light-directed chemical process. Each DNA chip
can contain, for example, thousands to millions of individual
synthetic polynucleotide probes arranged in a grid-like pattern and
miniaturized (e.g., to the size of a dime). Preferably, probes are
attached to a solid support in an ordered, addressable array.
A microarray can be composed of a large number of unique,
single-stranded polynucleotides, usually either synthetic antisense
polynucleotides or fragments of cDNAs, fixed to a solid support.
Typical polynucleotides are preferably about 6-60 nucleotides in
length, more preferably about 15-30 nucleotides in length, and most
preferably about 18-25 nucleotides in length. For certain types of
microarrays or other detection kits/systems, it may be preferable
to use oligonucleotides that are only about 7-20 nucleotides in
length. In other types of arrays, such as arrays used in
conjunction with chemiluminescent detection technology, preferred
probe lengths can be, for example, about 15-80 nucleotides in
length, preferably about 50-70 nucleotides in length, more
preferably about 55-65 nucleotides in length, and most preferably
about 60 nucleotides in length. The microarray or detection kit can
contain polynucleotides that cover the known 5' or 3' sequence of a
gene/transcript or target SNP site, sequential polynucleotides that
cover the full-length sequence of a gene/transcript; or unique
polynucleotides selected from particular areas along the length of
a target gene/transcript sequence, particularly areas corresponding
to one or more SNPs disclosed in Table 1 and/or Table 2.
Polynucleotides used in the microarray or detection kit can be
specific to a SNP or SNPs of interest (e.g., specific to a
particular SNP allele at a target SNP site, or specific to
particular SNP alleles at multiple different SNP sites), or
specific to a polymorphic gene/transcript or genes/transcripts of
interest.
Hybridization assays based on polynucleotide arrays rely on the
differences in hybridization stability of the probes to perfectly
matched and mismatched target sequence variants. For SNP
genotyping, it is generally preferable that stringency conditions
used in hybridization assays are high enough such that nucleic acid
molecules that differ from one another at as little as a single SNP
position can be differentiated (e.g., typical SNP hybridization
assays are designed so that hybridization will occur only if one
particular nucleotide is present at a SNP position, but will not
occur if an alternative nucleotide is present at that SNP
position). Such high stringency conditions may be preferable when
using, for example, nucleic acid arrays of allele-specific probes
for SNP detection. Such high stringency conditions are described in
the preceding section, and are well known to those skilled in the
art and can be found in, for example, Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6.
In other embodiments, the arrays are used in conjunction with
chemiluminescent detection technology. The following patents and
patent applications, which are all hereby incorporated by
reference, provide additional information pertaining to
chemiluminescent detection: U.S. patent application Ser. Nos.
10/620,332 and 10/620,333 describe chemiluminescent approaches for
microarray detection; U.S. Pat. Nos. 6,124,478, 6,107,024,
5,994,073, 5,981,768, 5,871,938, 5,843,681, 5,800,999, and
5,773,628 describe methods and compositions of dioxetane for
performing chemiluminescent detection; and U.S. published
application US2002/0110828 discloses methods and compositions for
microarray controls.
In one embodiment of the invention, a nucleic acid array can
comprise an array of probes of about 15-25 nucleotides in length.
In further embodiments, a nucleic acid array can comprise any
number of probes, in which at least one probe is capable of
detecting one or more SNPs disclosed in Table 1 and/or Table 2,
and/or at least one probe comprises a fragment of one of the
sequences selected from the group consisting of those disclosed in
Table 1, Table 2, the Sequence Listing, and sequences complementary
thereto, said fragment comprising at least about 8 consecutive
nucleotides, preferably 10, 12, 15, 16, 18, 20, more preferably 22,
25, 30, 40, 47, 50, 55, 60, 65, 70, 80, 90, 100, or more
consecutive nucleotides (or any other number in-between) and
containing (or being complementary to) a novel SNP allele disclosed
in Table 1 and/or Table 2. In some embodiments, the nucleotide
complementary to the SNP site is within 5, 4, 3, 2, or 1 nucleotide
from the center of the probe, more preferably at the center of said
probe.
A polynucleotide probe can be synthesized on the surface of the
substrate by using a chemical coupling procedure and an ink jet
application apparatus, as described in PCT application WO95/251116
(Baldeschweiler et al.) which is incorporated herein in its
entirety by reference. In another aspect, a "gridded" array
analogous to a dot (or slot) blot may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system, thermal, UV, mechanical or chemical bonding
procedures. An array, such as those described above, may be
produced by hand or by using available devices (slot blot or dot
blot apparatus), materials (any suitable solid support), and
machines (including robotic instruments), and may contain 8, 24,
96, 384, 1536, 6144 or more polynucleotides, or any other number
which lends itself to the efficient use of commercially available
instrumentation.
Using such arrays or other kits/systems, the present invention
provides methods of identifying the SNPs disclosed herein in a test
sample. Such methods typically involve incubating a test sample of
nucleic acids with an array comprising one or more probes
corresponding to at least one SNP position of the present
invention, and assaying for binding of a nucleic acid from the test
sample with one or more of the probes. Conditions for incubating a
SNP detection reagent (or a kit/system that employs one or more
such SNP detection reagents) with a test sample vary. Incubation
conditions depend on such factors as the format employed in the
assay, the detection methods employed, and the type and nature of
the detection reagents used in the assay. One skilled in the art
will recognize that any one of the commonly available
hybridization, amplification and array assay formats can readily be
adapted to detect the SNPs disclosed herein.
A SNP detection kit/system of the present invention may include
components that are used to prepare nucleic acids from a test
sample for the subsequent amplification and/or detection of a
SNP-containing nucleic acid molecule. Such sample preparation
components can be used to produce nucleic acid extracts (including
DNA and/or RNA), proteins or membrane extracts from any bodily
fluids (such as blood, serum, plasma, urine, saliva, phlegm,
gastric juices, semen, tears, sweat, etc.), skin, hair, cells
(especially nucleated cells), biopsies, buccal swabs or tissue
specimens. The test samples used in the above-described methods
will vary based on such factors as the assay format, nature of the
detection method, and the specific tissues, cells or extracts used
as the test sample to be assayed. Methods of preparing nucleic
acids, proteins, and cell extracts are well known in the art and
can be readily adapted to obtain a sample that is compatible with
the system utilized. Automated sample preparation systems for
extracting nucleic acids from a test sample are commercially
available, and examples are Qiagen's BioRobot 9600, Applied
Biosystems' PRISM 6700, and Roche Molecular Systems' COBAS
AmpliPrep System.
Another form of kit contemplated by the present invention is a
compartmentalized kit. A compartmentalized kit includes any kit in
which reagents are contained in separate containers. Such
containers include, for example, small glass containers, plastic
containers, strips of plastic, glass or paper, or arraying material
such as silica. Such containers allow one to efficiently transfer
reagents from one compartment to another compartment such that the
test samples and reagents are not cross-contaminated, or from one
container to another vessel not included in the kit, and the agents
or solutions of each container can be added in a quantitative
fashion from one compartment to another or to another vessel. Such
containers may include, for example, one or more containers which
will accept the test sample, one or more containers which contain
at least one probe or other SNP detection reagent for detecting one
or more SNPs of the present invention, one or more containers which
contain wash reagents (such as phosphate buffered saline,
Tris-buffers, etc.), and one or more containers which contain the
reagents used to reveal the presence of the bound probe or other
SNP detection reagents. The kit can optionally further comprise
compartments and/or reagents for, for example, nucleic acid
amplification or other enzymatic reactions such as primer extension
reactions, hybridization, ligation, electrophoresis (preferably
capillary electrophoresis), mass spectrometry, and/or laser-induced
fluorescent detection. The kit may also include instructions for
using the kit. Exemplary compartmentalized kits include
microfluidic devices known in the art (see, e.g., Weigl et al.,
"Lab-on-a-chip for drug development", Adv Drug Deliv Rev. 2003 Feb.
24; 55(3):349-77). In such microfluidic devices, the containers may
be referred to as, for example, microfluidic "compartments",
"chambers", or "channels".
Microfluidic devices, which may also be referred to as
"lab-on-a-chip" systems, biomedical micro-electro-mechanical
systems (bioMEMs), or multicomponent integrated systems, are
exemplary kits/systems of the present invention for analyzing SNPs.
Such systems miniaturize and compartmentalize processes such as
probe/target hybridization, nucleic acid amplification, and
capillary electrophoresis reactions in a single functional device.
Such microfluidic devices typically utilize detection reagents in
at least one aspect of the system, and such detection reagents may
be used to detect one or more SNPs of the present invention. One
example of a microfluidic system is disclosed in U.S. Pat. No.
5,589,136, which describes the integration of PCR amplification and
capillary electrophoresis in chips. Exemplary microfluidic systems
comprise a pattern of microchannels designed onto a glass, silicon,
quartz, or plastic wafer included on a microchip. The movements of
the samples may be controlled by electric, electroosmotic or
hydrostatic forces applied across different areas of the microchip
to create functional microscopic valves and pumps with no moving
parts. Varying the voltage can be used as a means to control the
liquid flow at intersections between the micro-machined channels
and to change the liquid flow rate for pumping across different
sections of the microchip. See, for example, U.S. Pat. No.
6,153,073, Dubrow et al., and U.S. Pat. No. 6,156,181, Parce et
al.
For genotyping SNPs, an exemplary microfluidic system may
integrate, for example, nucleic acid amplification, primer
extension, capillary electrophoresis, and a detection method such
as laser induced fluorescence detection. In a first step of an
exemplary process for using such an exemplary system, nucleic acid
samples are amplified, preferably by PCR. Then, the amplification
products are subjected to automated primer extension reactions
using ddNTPs (specific fluorescence for each ddNTP) and the
appropriate oligonucleotide primers to carry out primer extension
reactions which hybridize just upstream of the targeted SNP. Once
the extension at the 3' end is completed, the primers are separated
from the unincorporated fluorescent ddNTPs by capillary
electrophoresis. The separation medium used in capillary
electrophoresis can be, for example, polyacrylamide,
polyethyleneglycol or dextran. The incorporated ddNTPs in the
single nucleotide primer extension products are identified by
laser-induced fluorescence detection. Such an exemplary microchip
can be used to process, for example, at least 96 to 384 samples, or
more, in parallel.
Uses of Nucleic Acid Molecules
The nucleic acid molecules of the present invention have a variety
of uses, especially in the diagnosis and treatment of myocardial
infarction. For example, the nucleic acid molecules are useful as
hybridization probes, such as for genotyping SNPs in messenger RNA,
transcript, cDNA, genomic DNA, amplified DNA or other nucleic acid
molecules, and for isolating full-length cDNA and genomic clones
encoding the variant peptides disclosed in Table 1 as well as their
orthologs.
A probe can hybridize to any nucleotide sequence along the entire
length of a nucleic acid molecule provided in Table 1 and/or Table
2. Preferably, a probe of the present invention hybridizes to a
region of a target sequence that encompasses a SNP position
indicated in Table 1 and/or Table 2. More preferably, a probe
hybridizes to a SNP-containing target sequence in a
sequence-specific manner such that it distinguishes the target
sequence from other nucleotide sequences which vary from the target
sequence only by which nucleotide is present at the SNP site. Such
a probe is particularly useful for detecting the presence of a
SNP-containing nucleic acid in a test sample, or for determining
which nucleotide (allele) is present at a particular SNP site
(i.e., genotyping the SNP site).
A nucleic acid hybridization probe may be used for determining the
presence, level, form, and/or distribution of nucleic acid
expression. The nucleic acid whose level is determined can be DNA
or RNA. Accordingly, probes specific for the SNPs described herein
can be used to assess the presence, expression and/or gene copy
number in a given cell, tissue, or organism. These uses are
relevant for diagnosis of disorders involving an increase or
decrease in gene expression relative to normal levels. In vitro
techniques for detection of mRNA include, for example, Northern
blot hybridizations and in situ hybridizations. In vitro techniques
for detecting DNA include Southern blot hybridizations and in situ
hybridizations (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.).
Probes can be used as part of a diagnostic test kit for identifying
cells or tissues in which a variant protein is expressed, such as
by measuring the level of a variant protein-encoding nucleic acid
(e.g., mRNA) in a sample of cells from a subject or determining if
a polynucleotide contains a SNP of interest.
Thus, the nucleic acid molecules of the invention can be used as
hybridization probes to detect the SNPs disclosed herein, thereby
determining whether an individual with the polymorphisms is at risk
for myocardial infarction or has developed early stage myocardial
infarction. Detection of a SNP associated with a disease phenotype
provides a diagnostic tool for an active disease and/or genetic
predisposition to the disease.
The nucleic acid molecules of the invention are also useful as
primers to amplify any given region of a nucleic acid molecule,
particularly a region containing a SNP identified in Table 1 and/or
Table 2.
The nucleic acid molecules of the invention are also useful for
constructing recombinant vectors (described in greater detail
below). Such vectors include expression vectors that express a
portion of, or all of, any of the variant peptide sequences
provided in Table 1. Vectors also include insertion vectors, used
to integrate into another nucleic acid molecule sequence, such as
into the cellular genome, to alter in situ expression of a gene
and/or gene product. For example, an endogenous coding sequence can
be replaced via homologous recombination with all or part of the
coding region containing one or more specifically introduced
SNPs.
The nucleic acid molecules of the invention are also useful for
expressing antigenic portions of the variant proteins, particularly
antigenic portions that contain a variant amino acid sequence
(e.g., an amino acid substitution) caused by a SNP disclosed in
Table 1 and/or Table 2.
The nucleic acid molecules of the invention are also useful for
constructing vectors containing a gene regulatory region of the
nucleic acid molecules of the present invention.
The nucleic acid molecules of the invention are also useful for
designing ribozymes corresponding to all, or a part, of an mRNA
molecule expressed from a SNP-containing nucleic acid molecule
described herein.
The nucleic acid molecules of the invention are also useful for
constructing host cells expressing a part, or all, of the nucleic
acid molecules and variant peptides.
The nucleic acid molecules of the invention are also useful for
constructing transgenic animals expressing all, or a part, of the
nucleic acid molecules and variant peptides. The production of
recombinant cells and transgenic animals having nucleic acid
molecules which contain the SNPs disclosed in Table 1 and/or Table
2 allow, for example, effective clinical design of treatment
compounds and dosage regimens.
The nucleic acid molecules of the invention are also useful in
assays for drug screening to identify compounds that, for example,
modulate nucleic acid expression.
The nucleic acid molecules of the invention are also useful in gene
therapy in patients whose cells have aberrant gene expression.
Thus, recombinant cells, which include a patient's cells that have
been engineered ex vivo and returned to the patient, can be
introduced into an individual where the recombinant cells produce
the desired protein to treat the individual.
SNP Genotyping Methods
The process of determining which specific nucleotide (i.e., allele)
is present at each of one or more SNP positions, such as a SNP
position in a nucleic acid molecule disclosed in Table 1 and/or
Table 2, is referred to as SNP genotyping. The present invention
provides methods of SNP genotyping, such as for use in screening
for myocardial infarction or related pathologies, or determining
predisposition thereto, or determining responsiveness to a form of
treatment, or in genome mapping or SNP association analysis,
etc.
Nucleic acid samples can be genotyped to determine which allele(s)
is/are present at any given genetic region (e.g., SNP position) of
interest by methods well known in the art. The neighboring sequence
can be used to design SNP detection reagents such as
oligonucleotide probes, which may optionally be implemented in a
kit format. Exemplary SNP genotyping methods are described in Chen
et al., "Single nucleotide polymorphism genotyping: biochemistry,
protocol, cost and throughput", Pharmacogenomics J. 2003;
3(2):77-96; Kwok et al., "Detection of single nucleotide
polymorphisms", Curr Issues Mol. Biol. 2003 April; 5(2):43-60; Shi,
"Technologies for individual genotyping: detection of genetic
polymorphisms in drug targets and disease genes", Am J
Pharmacogenomics. 2002; 2(3):197-205; and Kwok, "Methods for
genotyping single nucleotide polymorphisms", Annu Rev Genomics Hum
Genet 2001; 2:235-58. Exemplary techniques for high-throughput SNP
genotyping are described in Marnellos, "High-throughput SNP
analysis for genetic association studies", Curr Opin Drug Discov
Devel. 2003 May; 6(3):317-21. Common SNP genotyping methods
include, but are not limited to, TaqMan assays, molecular beacon
assays, nucleic acid arrays, allele-specific primer extension,
allele-specific PCR, arrayed primer extension, homogeneous primer
extension assays, primer extension with detection by mass
spectrometry, pyrosequencing, multiplex primer extension sorted on
genetic arrays, ligation with rolling circle amplification,
homogeneous ligation, OLA (U.S. Pat. No. 4,988,167), multiplex
ligation reaction sorted on genetic arrays, restriction-fragment
length polymorphism, single base extension-tag assays, and the
Invader assay. Such methods may be used in combination with
detection mechanisms such as, for example, luminescence or
chemiluminescence detection, fluorescence detection, time-resolved
fluorescence detection, fluorescence resonance energy transfer,
fluorescence polarization, mass spectrometry, and electrical
detection.
Various methods for detecting polymorphisms include, but are not
limited to, methods in which protection from cleavage agents is
used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes
(Myers et al., Science 230:1242 (1985); Cotton et al., PNAS 85:4397
(1988); and Saleeba et al., Meth. Enzymol. 217:286-295 (1992)),
comparison of the electrophoretic mobility of variant and wild type
nucleic acid molecules (Orita et al., PNAS 86:2766 (1989); Cotton
et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet.
Anal. Tech. Appl. 9:73-79 (1992)), and assaying the movement of
polymorphic or wild-type fragments in polyacrylamide gels
containing a gradient of denaturant using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)).
Sequence variations at specific locations can also be assessed by
nuclease protection assays such as RNase and S1 protection or
chemical cleavage methods.
In a preferred embodiment, SNP genotyping is performed using the
TaqMan assay, which is also known as the 5' nuclease assay (U.S.
Pat. Nos. 5,210,015 and 5,538,848). The TaqMan assay detects the
accumulation of a specific amplified product during PCR. The TaqMan
assay utilizes an oligonucleotide probe labeled with a fluorescent
reporter dye and a quencher dye. The reporter dye is excited by
irradiation at an appropriate wavelength, it transfers energy to
the quencher dye in the same probe via a process called
fluorescence resonance energy transfer (FRET). When attached to the
probe, the excited reporter dye does not emit a signal. The
proximity of the quencher dye to the reporter dye in the intact
probe maintains a reduced fluorescence for the reporter. The
reporter dye and quencher dye may be at the 5' most and the 3' most
ends, respectively, or vice versa. Alternatively, the reporter dye
may be at the 5' or 3' most end while the quencher dye is attached
to an internal nucleotide, or vice versa. In yet another
embodiment, both the reporter and the quencher may be attached to
internal nucleotides at a distance from each other such that
fluorescence of the reporter is reduced.
During PCR, the 5' nuclease activity of DNA polymerase cleaves the
probe, thereby separating the reporter dye and the quencher dye and
resulting in increased fluorescence of the reporter. Accumulation
of PCR product is detected directly by monitoring the increase in
fluorescence of the reporter dye. The DNA polymerase cleaves the
probe between the reporter dye and the quencher dye only if the
probe hybridizes to the target SNP-containing template which is
amplified during PCR, and the probe is designed to hybridize to the
target SNP site only if a particular SNP allele is present.
Preferred TaqMan primer and probe sequences can readily be
determined using the SNP and associated nucleic acid sequence
information provided herein. A number of computer programs, such as
Primer Express (Applied Biosystems, Foster City, Calif.), can be
used to rapidly obtain optimal primer/probe sets. It will be
apparent to one of skill in the art that such primers and probes
for detecting the SNPs of the present invention are useful in
diagnostic assays for myocardial infarction and related
pathologies, and can be readily incorporated into a kit format. The
present invention also includes modifications of the Taqman assay
well known in the art such as the use of Molecular Beacon probes
(U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats
(U.S. Pat. Nos. 5,866,336 and 6,117,635).
Another preferred method for genotyping the SNPs of the present
invention is the use of two oligonucleotide probes in an OLA (see,
e.g., U.S. Pat. No. 4,988,617). In this method, one probe
hybridizes to a segment of a target nucleic acid with its 3' most
end aligned with the SNP site. A second probe hybridizes to an
adjacent segment of the target nucleic acid molecule directly 3' to
the first probe. The two juxtaposed probes hybridize to the target
nucleic acid molecule, and are ligated in the presence of a linking
agent such as a ligase if there is perfect complementarity between
the 3' most nucleotide of the first probe with the SNP site. If
there is a mismatch, ligation would not occur. After the reaction,
the ligated probes are separated from the target nucleic acid
molecule, and detected as indicators of the presence of a SNP.
The following patents, patent applications, and published
international patent applications, which are all hereby
incorporated by reference, provide additional information
pertaining to techniques for carrying out various types of OLA:
U.S. Pat. Nos. 6,027,889, 6,268,148, 5,494,810, 5,830,711, and
6,054,564 describe OLA strategies for performing SNP detection; WO
97/31256 and WO 00/56927 describe OLA strategies for performing SNP
detection using universal arrays, wherein a zipcode sequence can be
introduced into one of the hybridization probes, and the resulting
product, or amplified product, hybridized to a universal zip code
array; U.S. application US01/17329 (and Ser. No. 09/584,905)
describes OLA (or LDR) followed by PCR, wherein zipcodes are
incorporated into OLA probes, and amplified PCR products are
determined by electrophoretic or universal zipcode array readout;
U.S. application 60/427,818, 60/445,636, and 60/445,494 describe
SNPlex methods and software for multiplexed SNP detection using OLA
followed by PCR, wherein zipcodes are incorporated into OLA probes,
and amplified PCR products are hybridized with a zipchute reagent,
and the identity of the SNP determined from electrophoretic readout
of the zipchute. In some embodiments, OLA is carried out prior to
PCR (or another method of nucleic acid amplification). In other
embodiments, PCR (or another method of nucleic acid amplification)
is carried out prior to OLA.
Another method for SNP genotyping is based on mass spectrometry.
Mass spectrometry takes advantage of the unique mass of each of the
four nucleotides of DNA. SNPs can be unambiguously genotyped by
mass spectrometry by measuring the differences in the mass of
nucleic acids having alternative SNP alleles. MALDI-TOF (Matrix
Assisted Laser Desorption Ionization--Time of Flight) mass
spectrometry technology is preferred for extremely precise
determinations of molecular mass, such as SNPs. Numerous approaches
to SNP analysis have been developed based on mass spectrometry.
Preferred mass spectrometry-based methods of SNP genotyping include
primer extension assays, which can also be utilized in combination
with other approaches, such as traditional gel-based formats and
microarrays.
Typically, the primer extension assay involves designing and
annealing a primer to a template PCR amplicon upstream (5') from a
target SNP position. A mix of dideoxynucleotide triphosphates
(ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to
a reaction mixture containing template (e.g., a SNP-containing
nucleic acid molecule which has typically been amplified, such as
by PCR), primer, and DNA polymerase. Extension of the primer
terminates at the first position in the template where a nucleotide
complementary to one of the ddNTPs in the mix occurs. The primer
can be either immediately adjacent (i.e., the nucleotide at the 3'
end of the primer hybridizes to the nucleotide next to the target
SNP site) or two or more nucleotides removed from the SNP position.
If the primer is several nucleotides removed from the target SNP
position, the only limitation is that the template sequence between
the 3' end of the primer and the SNP position cannot contain a
nucleotide of the same type as the one to be detected, or this will
cause premature termination of the extension primer. Alternatively,
if all four ddNTPs alone, with no dNTPs, are added to the reaction
mixture, the primer will always be extended by only one nucleotide,
corresponding to the target SNP position. In this instance, primers
are designed to bind one nucleotide upstream from the SNP position
(i.e., the nucleotide at the 3' end of the primer hybridizes to the
nucleotide that is immediately adjacent to the target SNP site on
the 5' side of the target SNP site). Extension by only one
nucleotide is preferable, as it minimizes the overall mass of the
extended primer, thereby increasing the resolution of mass
differences between alternative SNP nucleotides. Furthermore,
mass-tagged ddNTPs can be employed in the primer extension
reactions in place of unmodified ddNTPs. This increases the mass
difference between primers extended with these ddNTPs, thereby
providing increased sensitivity and accuracy, and is particularly
useful for typing heterozygous base positions. Mass-tagging also
alleviates the need for intensive sample-preparation procedures and
decreases the necessary resolving power of the mass
spectrometer.
The extended primers can then be purified and analyzed by MALDI-TOF
mass spectrometry to determine the identity of the nucleotide
present at the target SNP position. In one method of analysis, the
products from the primer extension reaction are combined with light
absorbing crystals that form a matrix. The matrix is then hit with
an energy source such as a laser to ionize and desorb the nucleic
acid molecules into the gas-phase. The ionized molecules are then
ejected into a flight tube and accelerated down the tube towards a
detector. The time between the ionization event, such as a laser
pulse, and collision of the molecule with the detector is the time
of flight of that molecule. The time of flight is precisely
correlated with the mass-to-charge ratio (m/z) of the ionized
molecule. Ions with smaller m/z travel down the tube faster than
ions with larger m/z and therefore the lighter ions reach the
detector before the heavier ions. The time-of-flight is then
converted into a corresponding, and highly precise, m/z. In this
manner, SNPs can be identified based on the slight differences in
mass, and the corresponding time of flight differences, inherent in
nucleic acid molecules having different nucleotides at a single
base position. For further information regarding the use of primer
extension assays in conjunction with MALDI-TOF mass spectrometry
for SNP genotyping, see, e.g., Wise et al., "A standard protocol
for single nucleotide primer extension in the human genome using
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry", Rapid Commun Mass Spectrom. 2003;
17(11):1195-202.
The following references provide further information describing
mass spectrometry-based methods for SNP genotyping: Bocker, "SNP
and mutation discovery using base-specific cleavage and MALDI-TOF
mass spectrometry", Bioinformatics. 2003 July; 19 Suppl 1:I44-I53;
Storm et al., "MALDI-TOF mass spectrometry-based SNP genotyping",
Methods Mol. Biol. 2003; 212:241-62; Jurinke et al., "The use of
MassARRAY technology for high throughput genotyping", Adv Biochem
Eng Biotechnol. 2002; 77:57-74; and Jurinke et al., "Automated
genotyping using the DNA MassArray technology", Methods Mol. Biol.
2002; 187:179-92.
SNPs can also be scored by direct DNA sequencing. A variety of
automated sequencing procedures can be utilized ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO94/16101; Cohen et
al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl.
Biochem. Biotechnol. 38:147-159 (1993)). The nucleic acid sequences
of the present invention enable one of ordinary skill in the art to
readily design sequencing primers for such automated sequencing
procedures. Commercial instrumentation, such as the Applied
Biosystems 377, 3100, 3700, 3730, and 3730.times.1 DNA Analyzers
(Foster City, Calif.), is commonly used in the art for automated
sequencing.
Other methods that can be used to genotype the SNPs of the present
invention include single-strand conformational polymorphism (SSCP),
and denaturing gradient gel electrophoresis (DGGE) (Myers et al.,
Nature 313:495 (1985)). SSCP identifies base differences by
alteration in electrophoretic migration of single stranded PCR
products, as described in Orita et al., Proc. Nat. Acad.
Single-stranded PCR products can be generated by heating or
otherwise denaturing double stranded PCR products. Single-stranded
nucleic acids may refold or form secondary structures that are
partially dependent on the base sequence. The different
electrophoretic mobilities of single-stranded amplification
products are related to base-sequence differences at SNP positions.
DGGE differentiates SNP alleles based on the different
sequence-dependent stabilities and melting properties inherent in
polymorphic DNA and the corresponding differences in
electrophoretic migration patterns in a denaturing gradient gel
(Erlich, ed., PCR Technology, Principles and Applications for DNA
Amplification, W.H. Freeman and Co, New York, 1992, Chapter 7).
Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be
used to score SNPs based on the development or loss of a ribozyme
cleavage site. Perfectly matched sequences can be distinguished
from mismatched sequences by nuclease cleavage digestion assays or
by differences in melting temperature. If the SNP affects a
restriction enzyme cleavage site, the SNP can be identified by
alterations in restriction enzyme digestion patterns, and the
corresponding changes in nucleic acid fragment lengths determined
by gel electrophoresis
SNP genotyping can include the steps of, for example, collecting a
biological sample from a human subject (e.g., sample of tissues,
cells, fluids, secretions, etc.), isolating nucleic acids (e.g.,
genomic DNA, mRNA or both) from the cells of the sample, contacting
the nucleic acids with one or more primers which specifically
hybridize to a region of the isolated nucleic acid containing a
target SNP under conditions such that hybridization and
amplification of the target nucleic acid region occurs, and
determining the nucleotide present at the SNP position of interest,
or, in some assays, detecting the presence or absence of an
amplification product (assays can be designed so that hybridization
and/or amplification will only occur if a particular SNP allele is
present or absent). In some assays, the size of the amplification
product is detected and compared to the length of a control sample;
for example, deletions and insertions can be detected by a change
in size of the amplified product compared to a normal genotype.
SNP genotyping is useful for numerous practical applications, as
described below. Examples of such applications include, but are not
limited to, SNP-disease association analysis, disease
predisposition screening, disease diagnosis, disease prognosis,
disease progression monitoring, determining therapeutic strategies
based on an individual's genotype ("pharmacogenomics"), developing
therapeutic agents based on SNP genotypes associated with a disease
or likelihood of responding to a drug, stratifying a patient
population for clinical trial for a treatment regimen, predicting
the likelihood that an individual will experience toxic side
effects from a therapeutic agent, and human identification
applications such as forensics.
Analysis of Genetic Association Between SNPs and Phenotypic
Traits
SNP genotyping for disease diagnosis, disease predisposition
screening, disease prognosis, determining drug responsiveness
(pharmacogenomics), drug toxicity screening, and other uses
described herein, typically relies on initially establishing a
genetic association between one or more specific SNPs and the
particular phenotypic traits of interest.
Different study designs may be used for genetic association studies
(Modern Epidemiology, Lippincott Williams & Wilkins (1998),
609-622). Observational studies are most frequently carried out in
which the response of the patients is not interfered with. The
first type of observational study identifies a sample of persons in
whom the suspected cause of the disease is present and another
sample of persons in whom the suspected cause is absent, and then
the frequency of development of disease in the two samples is
compared. These sampled populations are called cohorts, and the
study is a prospective study. The other type of observational study
is case-control or a retrospective study. In typical case-control
studies, samples are collected from individuals with the phenotype
of interest (cases) such as certain manifestations of a disease,
and from individuals without the phenotype (controls) in a
population (target population) that conclusions are to be drawn
from. Then the possible causes of the disease are investigated
retrospectively. As the time and costs of collecting samples in
case-control studies are considerably less than those for
prospective studies, case-control studies are the more commonly
used study design in genetic association studies, at least during
the exploration and discovery stage.
In both types of observational studies, there may be potential
confounding factors that should be taken into consideration.
Confounding factors are those that are associated with both the
real cause(s) of the disease and the disease itself, and they
include demographic information such as age, gender, ethnicity as
well as environmental factors. When confounding factors are not
matched in cases and controls in a study, and are not controlled
properly, spurious association results can arise. If potential
confounding factors are identified, they should be controlled for
by analysis methods explained below.
In a genetic association study, the cause of interest to be tested
is a certain allele or a SNP or a combination of alleles or a
haplotype from several SNPs. Thus, tissue specimens (e.g., whole
blood) from the sampled individuals may be collected and genomic
DNA genotyped for the SNP(s) of interest. In addition to the
phenotypic trait of interest, other information such as demographic
(e.g., age, gender, ethnicity, etc.), clinical, and environmental
information that may influence the outcome of the trait can be
collected to further characterize and define the sample set. In
many cases, these factors are known to be associated with diseases
and/or SNP allele frequencies. There are likely gene-environment
and/or gene-gene interactions as well. Analysis methods to address
gene-environment and gene-gene interactions (for example, the
effects of the presence of both susceptibility alleles at two
different genes can be greater than the effects of the individual
alleles at two genes combined) are discussed below.
After all the relevant phenotypic and genotypic information has
been obtained, statistical analyses are carried out to determine if
there is any significant correlation between the presence of an
allele or a genotype with the phenotypic characteristics of an
individual. Preferably, data inspection and cleaning are first
performed before carrying out statistical tests for genetic
association. Epidemiological and clinical data of the samples can
be summarized by descriptive statistics with tables and graphs.
Data validation is preferably performed to check for data
completion, inconsistent entries, and outliers. Chi-squared tests
and t-tests (Wilcoxon rank-sum tests if distributions are not
normal) may then be used to check for significant differences
between cases and controls for discrete and continuous variables,
respectively. To ensure genotyping quality, Hardy-Weinberg
disequilibrium tests can be performed on cases and controls
separately. Significant deviation from Hardy-Weinberg equilibrium
(HWE) in both cases and controls for individual markers can be
indicative of genotyping errors. If HWE is violated in a majority
of markers, it is indicative of population substructure that should
be further investigated. Moreover, Hardy-Weinberg disequilibrium in
cases only can indicate genetic association of the markers with the
disease (Genetic Data Analysis, Weir B., Sinauer (1990)).
To test whether an allele of a single SNP is associated with the
case or control status of a phenotypic trait, one skilled in the
art can compare allele frequencies in cases and controls. Standard
chi-squared tests and Fisher exact tests can be carried out on a
2.times.2 table (2 SNP alleles.times.2 outcomes in the categorical
trait of interest). To test whether genotypes of a SNP are
associated, chi-squared tests can be carried out on a 3.times.2
table (3 genotypes.times.2 outcomes). Score tests are also carried
out for genotypic association to contrast the three genotypic
frequencies (major homozygotes, heterozygotes and minor
homozygotes) in cases and controls, and to look for trends using 3
different modes of inheritance, namely dominant (with contrast
coefficients 2, -1, -1), additive (with contrast coefficients 1, 0,
-1) and recessive (with contrast coefficients 1, 1, -2). Odds
ratios for minor versus major alleles, and odds ratios for
heterozygote and homozygote variants versus the wild type genotypes
are calculated with the desired confidence limits, usually 95%.
In order to control for confounders and to test for interaction and
effect modifiers, stratified analyses may be performed using
stratified factors that are likely to be confounding, including
demographic information such as age, ethnicity, and gender, or an
interacting element or effect modifier, such as a known major gene
(e.g., APOE for Alzheimer's disease or HLA genes for autoimmune
diseases), or environmental factors such as smoking in lung cancer.
Stratified association tests may be carried out using
Cochran-Mantel-Haenszel tests that take into account the ordinal
nature of genotypes with 0, 1, and 2 variant alleles. Exact tests
by StatXact may also be performed when computationally possible.
Another way to adjust for confounding effects and test for
interactions is to perform stepwise multiple logistic regression
analysis using statistical packages such as SAS or R. Logistic
regression is a model-building technique in which the best fitting
and most parsimonious model is built to describe the relation
between the dichotomous outcome (for instance, getting a certain
disease or not) and a set of independent variables (for instance,
genotypes of different associated genes, and the associated
demographic and environmental factors). The most common model is
one in which the logit transformation of the odds ratios is
expressed as a linear combination of the variables (main effects)
and their cross-product terms (interactions) (Applied Logistic
Regression, Hosmer and Lemeshow, Wiley (2000)). To test whether a
certain variable or interaction is significantly associated with
the outcome, coefficients in the model are first estimated and then
tested for statistical significance of their departure from
zero.
In addition to performing association tests one marker at a time,
haplotype association analysis may also be performed to study a
number of markers that are closely linked together. Haplotype
association tests can have better power than genotypic or allelic
association tests when the tested markers are not the
disease-causing mutations themselves but are in linkage
disequilibrium with such mutations. The test will even be more
powerful if the disease is indeed caused by a combination of
alleles on a haplotype (e.g., APOE is a haplotype formed by 2 SNPs
that are very close to each other). In order to perform haplotype
association effectively, marker-marker linkage disequilibrium
measures, both D' and R.sup.2, are typically calculated for the
markers within a gene to elucidate the haplotype structure. Recent
studies (Daly et al, Nature Genetics, 29, 232-235, 2001) in linkage
disequilibrium indicate that SNPs within a gene are organized in
block pattern, and a high degree of linkage disequilibrium exists
within blocks and very little linkage disequilibrium exists between
blocks. Haplotype association with the disease status can be
performed using such blocks once they have been elucidated.
Haplotype association tests can be carried out in a similar fashion
as the allelic and genotypic association tests. Each haplotype in a
gene is analogous to an allele in a multi-allelic marker. One
skilled in the art can either compare the haplotype frequencies in
cases and controls or test genetic association with different pairs
of haplotypes. It has been proposed (Schaid et al, Am. J. Hum.
Genet., 70, 425-434, 2002) that score tests can be done on
haplotypes using the program "haplo.score". In that method,
haplotypes are first inferred by EM algorithm and score tests are
carried out with a generalized linear model (GLM) framework that
allows the adjustment of other factors.
An important decision in the performance of genetic association
tests is the determination of the significance level at which
significant association can be declared when the p-value of the
tests reaches that level. In an exploratory analysis where positive
hits will be followed up in subsequent confirmatory testing, an
unadjusted p-value <0.1 (a significance level on the lenient
side) may be used for generating hypotheses for significant
association of a SNP with certain phenotypic characteristics of a
disease. It is preferred that a p-value <0.05 (a significance
level traditionally used in the art) is achieved in order for a SNP
to be considered to have an association with a disease. It is more
preferred that a p-value <0.01 (a significance level on the
stringent side) is achieved for an association to be declared. When
hits are followed up in confirmatory analyses in more samples of
the same source or in different samples from different sources,
adjustment for multiple testing will be performed as to avoid
excess number of hits while maintaining the experiment-wise error
rates at 0.05. While there are different methods to adjust for
multiple testing to control for different kinds of error rates, a
commonly used but rather conservative method is Bonferroni
correction to control the experiment-wise or family-wise error rate
(Multiple comparisons and multiple tests, Westfall et al, SAS
Institute (1999)). Permutation tests to control for the false
discovery rates, FDR, can be more powerful (Benjamini and Hochberg,
Journal of the Royal Statistical Society, Series B 57, 1289-1300,
1995, Resampling-based Multiple Testing, Westfall and Young, Wiley
(1993)). Such methods to control for multiplicity would be
preferred when the tests are dependent and controlling for false
discovery rates is sufficient as opposed to controlling for the
experiment-wise error rates.
In replication studies using samples from different populations
after statistically significant markers have been identified in the
exploratory stage, meta-analyses can then be performed by combining
evidence of different studies (Modern Epidemiology, Lippincott
Williams & Wilkins, 1998, 643-673). If available, association
results known in the art for the same SNPs can be included in the
meta-analyses.
Since both genotyping and disease status classification can involve
errors, sensitivity analyses may be performed to see how odds
ratios and p-values would change upon various estimates on
genotyping and disease classification error rates.
It has been well known that subpopulation-based sampling bias
between cases and controls can lead to spurious results in
case-control association studies (Ewens and Spielman, Am. J. Hum.
Genet. 62, 450-458, 1995) when prevalence of the disease is
associated with different subpopulation groups. Such bias can also
lead to a loss of statistical power in genetic association studies.
To detect population stratification, Pritchard and Rosenberg
(Pritchard et al. Am. J. Hum. Gen. 1999, 65:220-228) suggested
typing markers that are unlinked to the disease and using results
of association tests on those markers to determine whether there is
any population stratification. When stratification is detected, the
genomic control (GC) method as proposed by Devlin and Roeder
(Devlin et al. Biometrics 1999, 55:997-1004) can be used to adjust
for the inflation of test statistics due to population
stratification. GC method is robust to changes in population
structure levels as well as being applicable to DNA pooling designs
(Devlin et al. Genet. Epidem. 2001, 21:273-284).
While Pritchard's method recommended using 15-20 unlinked
microsatellite markers, it suggested using more than 30 biallelic
markers to get enough power to detect population stratification.
For the GC method, it has been shown (Bacanu et al. Am. J. Hum.
Genet. 2000, 66:1933-1944) that about 60-70 biallelic markers are
sufficient to estimate the inflation factor for the test statistics
due to population stratification. Hence, 70 intergenic SNPs can be
chosen in unlinked regions as indicated in a genome scan (Kehoe et
al. Hum. Mol. Genet. 1999, 8:237-245).
Once individual risk factors, genetic or non-genetic, have been
found for the predisposition to disease, the next step is to set up
a classification/prediction scheme to predict the category (for
instance, disease or no-disease) that an individual will be in
depending on his genotypes of associated SNPs and other non-genetic
risk factors. Logistic regression for discrete trait and linear
regression for continuous trait are standard techniques for such
tasks (Applied Regression Analysis, Draper and Smith, Wiley
(1998)). Moreover, other techniques can also be used for setting up
classification. Such techniques include, but are not limited to,
MART, CART, neural network, and discriminant analyses that are
suitable for use in comparing the performance of different methods
(The Elements of Statistical Learning, Hastie, Tibshirani &
Friedman, Springer (2002)).
Disease Diagnosis and Predisposition Screening
Information on association/correlation between genotypes and
disease-related phenotypes can be exploited in several ways. For
example, in the case of a highly statistically significant
association between one or more SNPs with predisposition to a
disease for which treatment is available, detection of such a
genotype pattern in an individual may justify immediate
administration of treatment, or at least the institution of regular
monitoring of the individual. Detection of the susceptibility
alleles associated with serious disease in a couple contemplating
having children may also be valuable to the couple in their
reproductive decisions. In the case of a weaker but still
statistically significant association between a SNP and a human
disease, immediate therapeutic intervention or monitoring may not
be justified after detecting the susceptibility allele or SNP.
Nevertheless, the subject can be motivated to begin simple
life-style changes (e.g., diet, exercise) that can be accomplished
at little or no cost to the individual but would confer potential
benefits in reducing the risk of developing conditions for which
that individual may have an increased risk by virtue of having the
susceptibility allele(s).
The SNPs of the invention may contribute to myocardial infarction
in an individual in different ways. Some polymorphisms occur within
a protein coding sequence and contribute to disease phenotype by
affecting protein structure. Other polymorphisms occur in noncoding
regions but may exert phenotypic effects indirectly via influence
on, for example, replication, transcription, and/or translation. A
single SNP may affect more than one phenotypic trait. Likewise, a
single phenotypic trait may be affected by multiple SNPs in
different genes.
As used herein, the terms "diagnose", "diagnosis", and
"diagnostics" include, but are not limited to any of the following:
detection of myocardial infarction that an individual may presently
have or be at risk for, predisposition screening (i.e., determining
the increased risk for an individual in developing myocardial
infarction in the future, or determining whether an individual has
a decreased risk of developing myocardial infarction in the future;
in the case of recurrent myocardial infarction (RMI),
predisposition screening may typically involve determining the risk
that an individual who has previously had a myocardial infarction
will develop another myocardial infarction in the future, or
determining whether an individual who has not experienced a
myocardial infarction will be at risk for developing recurrent
myocardial infarctions in the future), determining a particular
type or subclass of myocardial infarction in an individual known to
have myocardial infarction, confirming or reinforcing a previously
made diagnosis of myocardial infarction, pharmacogenomic evaluation
of an individual to determine which therapeutic strategy that
individual is most likely to positively respond to or to predict
whether a patient is likely to respond to a particular treatment,
predicting whether a patient is likely to experience toxic effects
from a particular treatment or therapeutic compound, and evaluating
the future prognosis of an individual having myocardial infarction.
Such diagnostic uses are based on the SNPs individually or in a
unique combination or SNP haplotypes of the present invention.
Haplotypes are particularly useful in that, for example, fewer SNPs
can be genotyped to determine if a particular genomic region
harbors a locus that influences a particular phenotype, such as in
linkage disequilibrium-based SNP association analysis.
Linkage disequilibrium (LD) refers to the co-inheritance of alleles
(e.g., alternative nucleotides) at two or more different SNP sites
at frequencies greater than would be expected from the separate
frequencies of occurrence of each allele in a given population. The
expected frequency of co-occurrence of two alleles that are
inherited independently is the frequency of the first allele
multiplied by the frequency of the second allele. Alleles that
co-occur at expected frequencies are said to be in "linkage
equilibrium". In contrast, LD refers to any non-random genetic
association between allele(s) at two or more different SNP sites,
which is generally due to the physical proximity of the two loci
along a chromosome. LD can occur when two or more SNPs sites are in
close physical proximity to each other on a given chromosome and
therefore alleles at these SNP sites will tend to remain
unseparated for multiple generations with the consequence that a
particular nucleotide (allele) at one SNP site will show a
non-random association with a particular nucleotide (allele) at a
different SNP site located nearby. Hence, genotyping one of the SNP
sites will give almost the same information as genotyping the other
SNP site that is in LD.
For diagnostic purposes, if a particular SNP site is found to be
useful for diagnosing myocardial infarction, then the skilled
artisan would recognize that other SNP sites which are in LD with
this SNP site would also be useful for diagnosing the condition.
Various degrees of LD can be encountered between two or more SNPs
with the result being that some SNPs are more closely associated
(i.e., in stronger LD) than others. Furthermore, the physical
distance over which LD extends along a chromosome differs between
different regions of the genome, and therefore the degree of
physical separation between two or more SNP sites necessary for LD
to occur can differ between different regions of the genome.
For diagnostic applications, polymorphisms (e.g., SNPs and/or
haplotypes) that are not the actual disease-causing (causative)
polymorphisms, but are in LD with such causative polymorphisms, are
also useful. In such instances, the genotype of the polymorphism(s)
that is/are in LD with the causative polymorphism is predictive of
the genotype of the causative polymorphism and, consequently,
predictive of the phenotype (e.g., myocardial infarction) that is
influenced by the causative SNP(s). Thus, polymorphic markers that
are in LD with causative polymorphisms are useful as diagnostic
markers, and are particularly useful when the actual causative
polymorphism(s) is/are unknown.
Linkage disequilibrium in the human genome is reviewed in: Wall et
al., "Haplotype blocks and linkage disequilibrium in the human
genome", Nat Rev Genet. 2003 August; 4(8):587-97; Garner et al.,
"On selecting markers for association studies: patterns of linkage
disequilibrium between two and three diallelic loci", Genet
Epidemiol. 2003 January; 24(1):57-67; Ardlie et al., "Patterns of
linkage disequilibrium in the human genome", Nat Rev Genet. 2002
April; 3(4):299-309 (erratum in Nat Rev Genet 2002 July; 3(7):566);
and Remm et al., "High-density genotyping and linkage
disequilibrium in the human genome using chromosome 22 as a model";
Curr Opin Chem Biol. 2002 February; 6(1):24-30.
The contribution or association of particular SNPs and/or SNP
haplotypes with disease phenotypes, such as myocardial infarction,
enables the SNPs of the present invention to be used to develop
superior diagnostic tests capable of identifying individuals who
express a detectable trait, such as myocardial infarction, as the
result of a specific genotype, or individuals whose genotype places
them at an increased or decreased risk of developing a detectable
trait at a subsequent time as compared to individuals who do not
have that genotype. As described herein, diagnostics may be based
on a single SNP or a group of SNPs. Combined detection of a
plurality of SNPs (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 30, 32, 48, 50, 64, 96,
100, or any other number in-between, or more, of the SNPs provided
in Table 1 and/or Table 2) typically increases the probability of
an accurate diagnosis. For example, the presence of a single SNP
known to correlate with myocardial infarction might indicate a
probability of 20% that an individual has or is at risk of
developing myocardial infarction, whereas detection of five SNPs,
each of which correlates with myocardial infarction, might indicate
a probability of 80% that an individual has or is at risk of
developing myocardial infarction. To further increase the accuracy
of diagnosis or predisposition screening, analysis of the SNPs of
the present invention can be combined with that of other
polymorphisms or other risk factors of myocardial infarction, such
as disease symptoms, pathological characteristics, family history,
diet, environmental factors or lifestyle factors.
It will, of course, be understood by practitioners skilled in the
treatment or diagnosis of myocardial infarction that the present
invention generally does not intend to provide an absolute
identification of individuals who are at risk (or less at risk) of
developing myocardial infarction, and/or pathologies related to
myocardial infarction, but rather to indicate a certain increased
(or decreased) degree or likelihood of developing the disease based
on statistically significant association results. However, this
information is extremely valuable as it can be used to, for
example, initiate preventive treatments or to allow an individual
carrying one or more significant SNPs or SNP haplotypes to foresee
warning signs such as minor clinical symptoms, or to have regularly
scheduled physical exams to monitor for appearance of a condition
in order to identify and begin treatment of the condition at an
early stage. Particularly with diseases that are extremely
debilitating or fatal if not treated on time, the knowledge of a
potential predisposition, even if this predisposition is not
absolute, would likely contribute in a very significant manner to
treatment efficacy.
The diagnostic techniques of the present invention may employ a
variety of methodologies to determine whether a test subject has a
SNP or a SNP pattern associated with an increased or decreased risk
of developing a detectable trait or whether the individual suffers
from a detectable trait as a result of a particular
polymorphism/mutation, including, for example, methods which enable
the analysis of individual chromosomes for haplotyping, family
studies, single sperm DNA analysis, or somatic hybrids. The trait
analyzed using the diagnostics of the invention may be any
detectable trait that is commonly observed in pathologies and
disorders related to myocardial infarction.
Another aspect of the present invention relates to a method of
determining whether an individual is at risk (or less at risk) of
developing one or more traits or whether an individual expresses
one or more traits as a consequence of possessing a particular
trait-causing or trait-influencing allele. These methods generally
involve obtaining a nucleic acid sample from an individual and
assaying the nucleic acid sample to determine which nucleotide(s)
is/are present at one or more SNP positions, wherein the assayed
nucleotide(s) is/are indicative of an increased or decreased risk
of developing the trait or indicative that the individual expresses
the trait as a result of possessing a particular trait-causing or
trait-influencing allele.
In another embodiment, the SNP detection reagents of the present
invention are used to determine whether an individual has one or
more SNP allele(s) affecting the level (e.g., the concentration of
mRNA or protein in a sample, etc.) or pattern (e.g., the kinetics
of expression, rate of decomposition, stability profile, Km, Vmax,
etc.) of gene expression (collectively, the "gene response" of a
cell or bodily fluid). Such a determination can be accomplished by
screening for mRNA or protein expression (e.g., by using nucleic
acid arrays, RT-PCR, TaqMan assays, or mass spectrometry),
identifying genes having altered expression in an individual,
genotyping SNPs disclosed in Table 1 and/or Table 2 that could
affect the expression of the genes having altered expression (e.g.,
SNPs that are in and/or around the gene(s) having altered
expression, SNPs in regulatory/control regions, SNPs in and/or
around other genes that are involved in pathways that could affect
the expression of the gene(s) having altered expression, or all
SNPs could be genotyped), and correlating SNP genotypes with
altered gene expression. In this manner, specific SNP alleles at
particular SNP sites can be identified that affect gene
expression.
Pharmacogenomics and Therapeutics/Drug Development
The present invention provides methods for assessing the
pharmacogenomics of a subject harboring particular SNP alleles or
haplotypes to a particular therapeutic agent or pharmaceutical
compound, or to a class of such compounds. Pharmacogenomics deals
with the roles which clinically significant hereditary variations
(e.g., SNPs) play in the response to drugs due to altered drug
disposition and/or abnormal action in affected persons. See, e.g.,
Roses, Nature 405, 857-865 (2000); Gould Rothberg, Nature
Biotechnology 19, 209-211 (2001); Eichelbaum, Clin. Exp. Pharmacol.
Physiol. 23(10-11):983-985 (1996); and Linder, Clin. Chem.
43(2):254-266 (1997). The clinical outcomes of these variations can
result in severe toxicity of therapeutic drugs in certain
individuals or therapeutic failure of drugs in certain individuals
as a result of individual variation in metabolism. Thus, the SNP
genotype of an individual can determine the way a therapeutic
compound acts on the body or the way the body metabolizes the
compound. For example, SNPs in drug metabolizing enzymes can affect
the activity of these enzymes, which in turn can affect both the
intensity and duration of drug action, as well as drug metabolism
and clearance.
The discovery of SNPs in drug metabolizing enzymes, drug
transporters, proteins for pharmaceutical agents, and other drug
targets has explained why some patients do not obtain the expected
drug effects, show an exaggerated drug effect, or experience
serious toxicity from standard drug dosages. SNPs can be expressed
in the phenotype of the extensive metabolizer and in the phenotype
of the poor metabolizer. Accordingly, SNPs may lead to allelic
variants of a protein in which one or more of the protein functions
in one population are different from those in another population.
SNPs and the encoded variant peptides thus provide targets to
ascertain a genetic predisposition that can affect treatment
modality. For example, in a ligand-based treatment, SNPs may give
rise to amino terminal extracellular domains and/or other
ligand-binding regions of a receptor that are more or less active
in ligand binding, thereby affecting subsequent protein activation.
Accordingly, ligand dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing particular SNP alleles or haplotypes.
As an alternative to genotyping, specific variant proteins
containing variant amino acid sequences encoded by alternative SNP
alleles could be identified. Thus, pharmacogenomic characterization
of an individual permits the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
uses based on the individual's SNP genotype, thereby enhancing and
optimizing the effectiveness of the therapy. Furthermore, the
production of recombinant cells and transgenic animals containing
particular SNPs/haplotypes allow effective clinical design and
testing of treatment compounds and dosage regimens. For example,
transgenic animals can be produced that differ only in specific SNP
alleles in a gene that is orthologous to a human disease
susceptibility gene.
Pharmacogenomic uses of the SNPs of the present invention provide
several significant advantages for patient care, particularly in
treating myocardial infarction. Pharmacogenomic characterization of
an individual, based on an individual's SNP genotype, can identify
those individuals unlikely to respond to treatment with a
particular medication and thereby allows physicians to avoid
prescribing the ineffective medication to those individuals. On the
other hand, SNP genotyping of an individual may enable physicians
to select the appropriate medication and dosage regimen that will
be most effective based on an individual's SNP genotype. This
information increases a physician's confidence in prescribing
medications and motivates patients to comply with their drug
regimens. Furthermore, pharmacogenomics may identify patients
predisposed to toxicity and adverse reactions to particular drugs
or drug dosages. Adverse drug reactions lead to more than 100,000
avoidable deaths per year in the United States alone and therefore
represent a significant cause of hospitalization and death, as well
as a significant economic burden on the healthcare system (Pfost
et. al., Trends in Biotechnology, August 2000.). Thus,
pharmacogenomics based on the SNPs disclosed herein has the
potential to both save lives and reduce healthcare costs
substantially.
Pharmacogenomics in general is discussed further in Rose et al.,
"Pharmacogenetic analysis of clinically relevant genetic
polymorphisms", Methods Mol Med. 2003; 85:225-37. Pharmacogenomics
as it relates to Alzheimer's disease and other neurodegenerative
disorders is discussed in Cacabelos, "Pharmacogenomics for the
treatment of dementia", Ann Med. 2002; 34(5):357-79, Maimone et
al., "Pharmacogenomics of neurodegenerative diseases", Eur J
Pharmacol. 2001 February 9; 413(1):11-29, and Poirier,
"Apolipoprotein E: a pharmacogenetic target for the treatment of
Alzheimer's disease", Mol Diagn. 1999 December; 4(4):335-41.
Pharmacogenomics as it relates to cardiovascular disorders is
discussed in Siest et al., "Pharmacogenomics of drugs affecting the
cardiovascular system", Clin Chem Lab Med. 2003 April; 41(4):590-9,
Mukherjee et al., "Pharmacogenomics in cardiovascular diseases",
Prog Cardiovasc Dis. 2002 May-June; 44(6):479-98, and Mooser et
al., "Cardiovascular pharmacogenetics in the SNP era", J Thromb
Haemost. 2003 July; 1(7):1398-402. Pharmacogenomics as it relates
to cancer is discussed in McLeod et al., "Cancer pharmacogenomics:
SNPs, chips, and the individual patient", Cancer Invest. 2003;
21(4):630-40 and Watters et al., "Cancer pharmacogenomics: current
and future applications", Biochim Biophys Acta. 2003 Mar. 17;
1603(2):99-111.
The SNPs of the present invention also can be used to identify
novel therapeutic targets for myocardial infarction. For example,
genes containing the disease-associated variants ("variant genes")
or their products, as well as genes or their products that are
directly or indirectly regulated by or interacting with these
variant genes or their products, can be targeted for the
development of therapeutics that, for example, treat the disease or
prevent or delay disease onset. The therapeutics may be composed
of, for example, small molecules, proteins, protein fragments or
peptides, antibodies, nucleic acids, or their derivatives or
mimetics which modulate the functions or levels of the target genes
or gene products.
The SNP-containing nucleic acid molecules disclosed herein, and
their complementary nucleic acid molecules, may be used as
antisense constructs to control gene expression in cells, tissues,
and organisms. Antisense technology is well established in the art
and extensively reviewed in Antisense Drug Technology: Principles,
Strategies, and Applications, Crooke (ed.), Marcel Dekker, Inc.:
New York (2001). An antisense nucleic acid molecule is generally
designed to be complementary to a region of mRNA expressed by a
gene so that the antisense molecule hybridizes to the mRNA and
thereby blocks translation of mRNA into protein. Various classes of
antisense oligonucleouides are used in the art, two of which are
cleavers and blockers. Cleavers, by binding to target RNAs,
activate intracellular nucleases (e.g., RNaseH or RNase L) that
cleave the target RNA. Blockers, which also bind to target RNAs,
inhibit protein translation through steric hindrance of ribosomes.
Exemplary blockers include peptide nucleic acids, morpholinos,
locked nucleic acids, and methylphosphonates (see, e.g., Thompson,
Drug Discovery Today, 7 (17):912-917 (2002)). Antisense
oligonucleotides are directly useful as therapeutic agents, and are
also useful for determining and validating gene function (e.g., in
gene knock-out or knock-down experiments).
Antisense technology is further reviewed in: Lavery et al.,
"Antisense and RNAi: powerful tools in drug target discovery and
validation", Curr Opin Drug Discov Devel. 2003 July; 6(4):561-9;
Stephens et al., "Antisense oligonucleotide therapy in cancer",
Curr Opin Mol Ther. 2003 April; 5(2): 118-22; Kurreck, "Antisense
technologies. Improvement through novel chemical modifications",
Eur J. Biochem. 2003 April; 270(8):1628-44; Dias et al., "Antisense
oligonucleotides: basic concepts and mechanisms", Mol Cancer Ther.
2002 March; 1(5):347-55; Chen, "Clinical development of antisense
oligonucleotides as anti-cancer therapeutics", Methods Mol Med.
2003; 75:621-36; Wang et al., "Antisense anticancer oligonucleotide
therapeutics", Curr Cancer Drug Targets. 2001 November;
1(3):177-96; and Bennett, "Efficiency of antisense oligonucleotide
drug discovery", Antisense Nucleic Acid Drug Dev. 2002 June;
12(3):215-24.
The SNPs of the present invention are particularly useful for
designing antisense reagents that are specific for particular
nucleic acid variants. Based on the SNP information disclosed
herein, antisense oligonucleotides can be produced that
specifically target mRNA molecules that contain one or more
particular SNP nucleotides. In this manner, expression of mRNA
molecules that contain one or more undesired polymorphisms (e.g.,
SNP nucleotides that lead to a defective protein such as an amino
acid substitution in a catalytic domain) can be inhibited or
completely blocked. Thus, antisense oligonucleotides can be used to
specifically bind a particular polymorphic form (e.g., a SNP allele
that encodes a defective protein), thereby inhibiting translation
of this form, but which do not bind an alternative polymorphic form
(e.g., an alternative SNP nucleotide that encodes a protein having
normal function).
Antisense molecules can be used to inactivate mRNA in order to
inhibit gene expression and production of defective proteins.
Accordingly, these molecules can be used to treat a disorder, such
as myocardial infarction, characterized by abnormal or undesired
gene expression or expression of certain defective proteins. This
technique can involve cleavage by means of ribozymes containing
nucleotide sequences complementary to one or more regions in the
mRNA that attenuate the ability of the mRNA to be translated.
Possible mRNA regions include, for example, protein-coding regions
and particularly protein-coding regions corresponding to catalytic
activities, substrate/ligand binding, or other functional
activities of a protein.
The SNPs of the present invention are also useful for designing RNA
interference reagents that specifically target nucleic acid
molecules having particular SNP variants. RNA interference (RNAi),
also referred to as gene silencing, is based on using
double-stranded RNA (dsRNA) molecules to turn genes off. When
introduced into a cell, dsRNAs are processed by the cell into short
fragments (generally about 21-22 bp in length) known as small
interfering RNAs (siRNAs) which the cell uses in a
sequence-specific manner to recognize and destroy complementary
RNAs (Thompson, Drug Discovery Today, 7 (17): 912-917 (2002)).
Thus, because RNAi molecules, including siRNAs, act in a
sequence-specific manner, the SNPs of the present invention can be
used to design RNAi reagents that recognize and destroy nucleic
acid molecules having specific SNP alleles/nucleotides (such as
deleterious alleles that lead to the production of defective
proteins), while not affecting nucleic acid molecules having
alternative SNP alleles (such as alleles that encode proteins
having normal function). As with antisense reagents, RNAi reagents
may be directly useful as therapeutic agents (e.g., for turning off
defective, disease-causing genes), and are also useful for
characterizing and validating gene function (e.g., in gene
knock-out or knock-down experiments).
The following references provide a further review of RNAi: Agami,
"RNAi and related mechanisms and their potential use for therapy",
Curr Opin Chem Biol. 2002 December; 6(6):829-34; Lavery et al.,
"Antisense and RNAi: powerful tools in drug target discovery and
validation", Curr Opin Drug Discov Devel. 2003 July; 6(4):561-9;
Shi, "Mammalian RNAi for the masses", Trends Genet 2003 January;
19(1):9-12), Shuey et al., "RNAi: gene-silencing in therapeutic
intervention", Drug Discovery Today 2002 October; 7(20):1040-1046;
McManus et al., Nat Rev Genet 2002 October; 3(10):737-47; Xia et
al., Nat Biotechnol 2002 October; 20(10):1006-10; Plasterk et al,
Curr Opin Genet Dev 2000 October; 10(5):562-7; Bosher et al., Nat
Cell Biol 2000 February; 2(2):E31-6; and Hunter, Curr Biol 1999
Jun. 17; 9(12):R440-2).
A subject suffering from a pathological condition, such as
myocardial infarction, ascribed to a SNP may be treated so as to
correct the genetic defect (see Kren et al., Proc. Natl. Acad. Sci.
USA 96:10349-10354 (1999)). Such a subject can be identified by any
method that can detect the polymorphism in a biological sample
drawn from the subject. Such a genetic defect may be permanently
corrected by administering to such a subject a nucleic acid
fragment incorporating a repair sequence that supplies the
normal/wild-type nucleotide at the position of the SNP. This
site-specific repair sequence can encompass an RNA/DNA
oligonucleotide that operates to promote endogenous repair of a
subject's genomic DNA. The site-specific repair sequence is
administered in an appropriate vehicle, such as a complex with
polyethylenimine, encapsulated in anionic liposomes, a viral vector
such as an adenovirus, or other pharmaceutical composition that
promotes intracellular uptake of the administered nucleic acid. A
genetic defect leading to an inborn pathology may then be overcome,
as the chimeric oligonucleotides induce incorporation of the normal
sequence into the subject's genome. Upon incorporation, the normal
gene product is expressed, and the replacement is propagated,
thereby engendering a permanent repair and therapeutic enhancement
of the clinical condition of the subject.
In cases in which a cSNP results in a variant protein that is
ascribed to be the cause of, or a contributing factor to, a
pathological condition, a method of treating such a condition can
include administering to a subject experiencing the pathology the
wild-type/normal cognate of the variant protein. Once administered
in an effective dosing regimen, the wild-type cognate provides
complementation or remediation of the pathological condition.
The invention further provides a method for identifying a compound
or agent that can be used to treat myocardial infarction. The SNPs
disclosed herein are useful as targets for the identification
and/or development of therapeutic agents. A method for identifying
a therapeutic agent or compound typically includes assaying the
ability of the agent or compound to modulate the activity and/or
expression of a SNP-containing nucleic acid or the encoded product
and thus identifying an agent or a compound that can be used to
treat a disorder characterized by undesired activity or expression
of the SNP-containing nucleic acid or the encoded product. The
assays can be performed in cell-based and cell-free systems.
Cell-based assays can include cells naturally expressing the
nucleic acid molecules of interest or recombinant cells genetically
engineered to express certain nucleic acid molecules.
Variant gene expression in a myocardial infarction patient can
include, for example, either expression of a SNP-containing nucleic
acid sequence (for instance, a gene that contains a SNP can be
transcribed into an mRNA transcript molecule containing the SNP,
which can in turn be translated into a variant protein) or altered
expression of a normal/wild-type nucleic acid sequence due to one
or more SNPs (for instance, a regulatory/control region can contain
a SNP that affects the level or pattern of expression of a normal
transcript).
Assays for variant gene expression can involve direct assays of
nucleic acid levels (e.g., mRNA levels), expressed protein levels,
or of collateral compounds involved in a signal pathway. Further,
the expression of genes that are up- or down-regulated in response
to the signal pathway can also be assayed. In this embodiment, the
regulatory regions of these genes can be operably linked to a
reporter gene such as luciferase.
Modulators of variant gene expression can be identified in a method
wherein, for example, a cell is contacted with a candidate
compound/agent and the expression of mRNA determined. The level of
expression of mRNA in the presence of the candidate compound is
compared to the level of expression of mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of variant gene expression based on this comparison
and be used to treat a disorder such as myocardial infarction that
is characterized by variant gene expression (e.g., either
expression of a SNP-containing nucleic acid or altered expression
of a normal/wild-type nucleic acid molecule due to one or more SNPs
that affect expression of the nucleic acid molecule) due to one or
more SNPs of the present invention. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
The invention further provides methods of treatment, with the SNP
or associated nucleic acid domain (e.g., catalytic domain,
ligand/substrate-binding domain, regulatory/control region, etc.)
or gene, or the encoded mRNA transcript, as a target, using a
compound identified through drug screening as a gene modulator to
modulate variant nucleic acid expression. Modulation can include
either up-regulation (i.e., activation or agonization) or
down-regulation (i.e., suppression or antagonization) of nucleic
acid expression.
Expression of mRNA transcripts and encoded proteins, either wild
type or variant, may be altered in individuals with a particular
SNP allele in a regulatory/control element, such as a promoter or
transcription factor binding domain, that regulates expression. In
this situation, methods of treatment and compounds can be
identified, as discussed herein, that regulate or overcome the
variant regulatory/control element, thereby generating normal, or
healthy, expression levels of either the wild type or variant
protein.
The SNP-containing nucleic acid molecules of the present invention
are also useful for monitoring the effectiveness of modulating
compounds on the expression or activity of a variant gene, or
encoded product, in clinical trials or in a treatment regimen.
Thus, the gene expression pattern can serve as an indicator for the
continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance, as well as an indicator for toxicities. The gene
expression pattern can also serve as a marker indicative of a
physiological response of the affected cells to the compound.
Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
In another aspect of the present invention, there is provided a
pharmaceutical pack comprising a therapeutic agent (e.g., a small
molecule drug, antibody, peptide, antisense or RNAi nucleic acid
molecule, etc.) and a set of instructions for administration of the
therapeutic agent to humans diagnostically tested for one or more
SNPs or SNP haplotypes provided by the present invention.
The SNPs/haplotypes of the present invention are also useful for
improving many different aspects of the drug development process.
For example, individuals can be selected for clinical trials based
on their SNP genotype. Individuals with SNP genotypes that indicate
that they are most likely to respond to the drug can be included in
the trials and those individuals whose SNP genotypes indicate that
they are less likely to or would not respond to the drug, or suffer
adverse reactions, can be eliminated from the clinical trials. This
not only improves the safety of clinical trials, but also will
enhance the chances that the trial will demonstrate statistically
significant efficacy. Furthermore, the SNPs of the present
invention may explain why certain previously developed drugs
performed poorly in clinical trials and may help identify a subset
of the population that would benefit from a drug that had
previously performed poorly in clinical trials, thereby "rescuing"
previously developed drugs, and enabling the drug to be made
available to a particular myocardial infarction patient population
that can benefit from it.
SNPs have many important uses in drug discovery, screening, and
development. A high probability exists that, for any gene/protein
selected as a potential drug target, variants of that gene/protein
will exist in a patient population. Thus, determining the impact of
gene/protein variants on the selection and delivery of a
therapeutic agent should be an integral aspect of the drug
discovery and development process. (Jazwinska, A Trends Guide to
Genetic Variation and Genomic Medicine, 2002 March; S30-S36).
Knowledge of variants (e.g., SNPs and any corresponding amino acid
polymorphisms) of a particular therapeutic target (e.g., a gene,
mRNA transcript, or protein) enables parallel screening of the
variants in order to identify therapeutic candidates (e.g., small
molecule compounds, antibodies, antisense or RNAi nucleic acid
compounds, etc.) that demonstrate efficacy across variants
(Rothberg, Nat Biotechnol 2001 March; 19(3):209-11). Such
therapeutic candidates would be expected to show equal efficacy
across a larger segment of the patient population, thereby leading
to a larger potential market for the therapeutic candidate.
Furthermore, identifying variants of a potential therapeutic target
enables the most common form of the target to be used for selection
of therapeutic candidates, thereby helping to ensure that the
experimental activity that is observed for the selected candidates
reflects the real activity expected in the largest proportion of a
patient population (Jazwinska, A Trends Guide to Genetic Variation
and Genomic Medicine, 2002 March; S30-S36).
Additionally, screening therapeutic candidates against all known
variants of a target can enable the early identification of
potential toxicities and adverse reactions relating to particular
variants. For example, variability in drug absorption,
distribution, metabolism and excretion (ADME) caused by, for
example, SNPs in therapeutic targets or drug metabolizing genes,
can be identified, and this information can be utilized during the
drug development process to minimize variability in drug
disposition and develop therapeutic agents that are safer across a
wider range of a patient population. The SNPs of the present
invention, including the variant proteins and encoding polymorphic
nucleic acid molecules provided in Tables 1-2, are useful in
conjunction with a variety of toxicology methods established in the
art, such as those set forth in Current Protocols in Toxicology,
John Wiley & Sons, Inc., N.Y.
Furthermore, therapeutic agents that target any art-known proteins
(or nucleic acid molecules, either RNA or DNA) may cross-react with
the variant proteins (or polymorphic nucleic acid molecules)
disclosed in Table 1, thereby significantly affecting the
pharmacokinetic properties of the drug. Consequently, the protein
variants and the SNP-containing nucleic acid molecules disclosed in
Tables 1-2 are useful in developing, screening, and evaluating
therapeutic agents that target corresponding art-known protein
forms (or nucleic acid molecules). Additionally, as discussed
above, knowledge of all polymorphic forms of a particular drug
target enables the design of therapeutic agents that are effective
against most or all such polymorphic forms of the drug target.
Pharmaceutical Compositions and Administration Thereof
Any of the myocardial infarction-associated proteins, and encoding
nucleic acid molecules, disclosed herein can be used as therapeutic
targets (or directly used themselves as therapeutic compounds) for
treating myocardial infarction and related pathologies, and the
present disclosure enables therapeutic compounds (e.g., small
molecules, antibodies, therapeutic proteins, RNAi and antisense
molecules, etc.) to be developed that target (or are comprised of)
any of these therapeutic targets.
In general, a therapeutic compound will be administered in a
therapeutically effective amount by any of the accepted modes of
administration for agents that serve similar utilities. The actual
amount of the therapeutic compound of this invention, i.e., the
active ingredient, will depend upon numerous factors such as the
severity of the disease to be treated, the age and relative health
of the subject, the potency of the compound used, the route and
form of administration, and other factors.
Therapeutically effective amounts of therapeutic compounds may
range from, for example, approximately 0.01-50 mg per kilogram body
weight of the recipient per day; preferably about 0.1-20 mg/kg/day.
Thus, as an example, for administration to a 70 kg person, the
dosage range would most preferably be about 7 mg to 1.4 g per
day.
In general, therapeutic compounds will be administered as
pharmaceutical compositions by any one of the following routes:
oral, systemic (e.g., transdermal, intranasal, or by suppository),
or parenteral (e.g., intramuscular, intravenous, or subcutaneous)
administration. The preferred manner of administration is oral or
parenteral using a convenient daily dosage regimen, which can be
adjusted according to the degree of affliction. Oral compositions
can take the form of tablets, pills, capsules, semisolids, powders,
sustained release formulations, solutions, suspensions, elixirs,
aerosols, or any other appropriate compositions.
The choice of formulation depends on various factors such as the
mode of drug administration (e.g., for oral administration,
formulations in the form of tablets, pills, or capsules are
preferred) and the bioavailability of the drug substance. Recently,
pharmaceutical formulations have been developed especially for
drugs that show poor bioavailability based upon the principle that
bioavailability can be increased by increasing the surface area,
i.e., decreasing particle size. For example, U.S. Pat. No.
4,107,288 describes a pharmaceutical formulation having particles
in the size range from 10 to 1,000 nm in which the active material
is supported on a cross-linked matrix of macromolecules. U.S. Pat.
No. 5,145,684 describes the production of a pharmaceutical
formulation in which the drug substance is pulverized to
nanoparticles (average particle size of 400 nm) in the presence of
a surface modifier and then dispersed in a liquid medium to give a
pharmaceutical formulation that exhibits remarkably high
bioavailability.
Pharmaceutical compositions are comprised of, in general, a
therapeutic compound in combination with at least one
pharmaceutically acceptable excipient. Acceptable excipients are
non-toxic, aid administration, and do not adversely affect the
therapeutic benefit of the therapeutic compound. Such excipients
may be any solid, liquid, semi-solid or, in the case of an aerosol
composition, gaseous excipient that is generally available to one
skilled in the art.
Solid pharmaceutical excipients include starch, cellulose, talc,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk and the like. Liquid
and semisolid excipients may be selected from glycerol, propylene
glycol, water, ethanol and various oils, including those of
petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,
soybean oil, mineral oil, sesame oil, etc. Preferred liquid
carriers, particularly for injectable solutions, include water,
saline, aqueous dextrose, and glycols.
Compressed gases may be used to disperse a compound of this
invention in aerosol form. Inert gases suitable for this purpose
are nitrogen, carbon dioxide, etc.
Other suitable pharmaceutical excipients and their formulations are
described in Remington's Pharmaceutical Sciences, edited by E. W.
Martin (Mack Publishing Company, 18th ed., 1990).
The amount of the therapeutic compound in a formulation can vary
within the full range employed by those skilled in the art.
Typically, the formulation will contain, on a weight percent (wt %)
basis, from about 0.01-99.99 wt % of the therapeutic compound based
on the total formulation, with the balance being one or more
suitable pharmaceutical excipients. Preferably, the compound is
present at a level of about 1-80 wt %.
Therapeutic compounds can be administered alone or in combination
with other therapeutic compounds or in combination with one or more
other active ingredient(s). For example, an inhibitor or stimulator
of a myocardial infarction-associated protein can be administered
in combination with another agent that inhibits or stimulates the
activity of the same or a different myocardial
infarction-associated protein to thereby counteract the affects of
myocardial infarction.
For further information regarding pharmacology, see Current
Protocols in Pharmacology, John Wiley & Sons, Inc., N.Y.
Human Identification Applications
In addition to their diagnostic and therapeutic uses in myocardial
infarction and related pathologies, the SNPs provided by the
present invention are also useful as human identification markers
for such applications as forensics, paternity testing, and
biometrics (see, e.g., Gill, "An assessment of the utility of
single nucleotide polymorphisms (SNPs) for forensic purposes", Int
J Legal Med. 2001; 114(4-5):204-10). Genetic variations in the
nucleic acid sequences between individuals can be used as genetic
markers to identify individuals and to associate a biological
sample with an individual. Determination of which nucleotides
occupy a set of SNP positions in an individual identifies a set of
SNP markers that distinguishes the individual. The more SNP
positions that are analyzed, the lower the probability that the set
of SNPs in one individual is the same as that in an unrelated
individual. Preferably, if multiple sites are analyzed, the sites
are unlinked (i.e., inherited independently). Thus, preferred sets
of SNPs can be selected from among the SNPs disclosed herein, which
may include SNPs on different chromosomes, SNPs on different
chromosome arms, and/or SNPs that are dispersed over substantial
distances along the same chromosome arm.
Furthermore, among the SNPs disclosed herein, preferred SNPs for
use in certain forensic/human identification applications include
SNPs located at degenerate codon positions (i.e., the third
position in certain codons which can be one of two or more
alternative nucleotides and still encode the same amino acid),
since these SNPs do not affect the encoded protein. SNPs that do
not affect the encoded protein are expected to be under less
selective pressure and are therefore expected to be more
polymorphic in a population, which is typically an advantage for
forensic/human identification applications. However, for certain
forensics/human identification applications, such as predicting
phenotypic characteristics (e.g., inferring ancestry or inferring
one or more physical characteristics of an individual) from a DNA
sample, it may be desirable to utilize SNPs that affect the encoded
protein.
For many of the SNPs disclosed in Tables 1-2 (which are identified
as "Applera" SNP source), Tables 1-2 provide SNP allele frequencies
obtained by re-sequencing the DNA of chromosomes from 39
individuals (Tables 1-2 also provide allele frequency information
for "Celera" source SNPs and, where available, public SNPs from
dbEST, HGBASE, and/or HGMD). The allele frequencies provided in
Tables 1-2 enable these SNPs to be readily used for human
identification applications. Although any SNP disclosed in Table 1
and/or Table 2 could be used for human identification, the closer
that the frequency of the minor allele at a particular SNP site is
to 50%, the greater the ability of that SNP to discriminate between
different individuals in a population since it becomes increasingly
likely that two randomly selected individuals would have different
alleles at that SNP site. Using the SNP allele frequencies provided
in Tables 1-2, one of ordinary skill in the art could readily
select a subset of SNPs for which the frequency of the minor allele
is, for example, at least 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 45%,
or 50%, or any other frequency in-between. Thus, since Tables 1-2
provide allele frequencies based on the re-sequencing of the
chromosomes from 39 individuals, a subset of SNPs could readily be
selected for human identification in which the total allele count
of the minor allele at a particular SNP site is, for example, at
least 1, 2, 4, 8, 10, 16, 20, 24, 30, 32, 36, 38, 39, 40, or any
other number in-between.
Furthermore, Tables 1-2 also provide population group
(interchangeably referred to herein as ethnic or racial groups)
information coupled with the extensive allele frequency
information. For example, the group of 39 individuals whose DNA was
re-sequenced was made-up of 20 Caucasians and 19 African-Americans.
This population group information enables further refinement of SNP
selection for human identification. For example, preferred SNPs for
human identification can be selected from Tables 1-2 that have
similar allele frequencies in both the Caucasian and
African-American populations; thus, for example, SNPs can be
selected that have equally high discriminatory power in both
populations. Alternatively, SNPs can be selected for which there is
a statistically significant difference in allele frequencies
between the Caucasian and African-American populations (as an
extreme example, a particular allele may be observed only in either
the Caucasian or the African-American population group but not
observed in the other population group); such SNPs are useful, for
example, for predicting the race/ethnicity of an unknown
perpetrator from a biological sample such as a hair or blood stain
recovered at a crime scene. For a discussion of using SNPs to
predict ancestry from a DNA sample, including statistical methods,
see Frudakis et al., "A Classifier for the SNP-Based Inference of
Ancestry", Journal of Forensic Sciences 2003; 48(4):771-782.
SNPs have numerous advantages over other types of polymorphic
markers, such as short tandem repeats (STRs). For example, SNPs can
be easily scored and are amenable to automation, making SNPs the
markers of choice for large-scale forensic databases. SNPs are
found in much greater abundance throughout the genome than repeat
polymorphisms. Population frequencies of two polymorphic forms can
usually be determined with greater accuracy than those of multiple
polymorphic forms at multi-allelic loci. SNPs are mutationaly more
stable than repeat polymorphisms. SNPs are not susceptible to
artefacts such as stutter bands that can hinder analysis. Stutter
bands are frequently encountered when analyzing repeat
polymorphisms, and are particularly troublesome when analyzing
samples such as crime scene samples that may contain mixtures of
DNA from multiple sources. Another significant advantage of SNP
markers over STR markers is the much shorter length of nucleic acid
needed to score a SNP. For example, STR markers are generally
several hundred base pairs in length. A SNP, on the other hand,
comprises a single nucleotide, and generally a short conserved
region on either side of the SNP position for primer and/or probe
binding. This makes SNPs more amenable to typing in highly degraded
or aged biological samples that are frequently encountered in
forensic casework in which DNA may be fragmented into short
pieces.
SNPs also are not subject to microvariant and "off-ladder" alleles
frequently encountered when analyzing STR loci. Microvariants are
deletions or insertions within a repeat unit that change the size
of the amplified DNA product so that the amplified product does not
migrate at the same rate as reference alleles with normal sized
repeat units. When separated by size, such as by electrophoresis on
a polyacrylamide gel, microvariants do not align with a reference
allelic ladder of standard sized repeat units, but rather migrate
between the reference alleles. The reference allelic ladder is used
for precise sizing of alleles for allele classification; therefore
alleles that do not align with the reference allelic ladder lead to
substantial analysis problems. Furthermore, when analyzing
multi-allelic repeat polymorphisms, occasionally an allele is found
that consists of more or less repeat units than has been previously
seen in the population, or more or less repeat alleles than are
included in a reference allelic ladder. These alleles will migrate
outside the size range of known alleles in a reference allelic
ladder, and therefore are referred to as "off-ladder" alleles. In
extreme cases, the allele may contain so few or so many repeats
that it migrates well out of the range of the reference allelic
ladder. In this situation, the allele may not even be observed, or,
with multiplex analysis, it may migrate within or close to the size
range for another locus, further confounding analysis.
SNP analysis avoids the problems of microvariants and off-ladder
alleles encountered in STR analysis. Importantly, microvariants and
off-ladder alleles may provide significant problems, and may be
completely missed, when using analysis methods such as
oligonucleotide hybridization arrays, which utilize oligonucleotide
probes specific for certain known alleles. Furthermore, off-ladder
alleles and microvariants encountered with STR analysis, even when
correctly typed, may lead to improper statistical analysis, since
their frequencies in the population are generally unknown or poorly
characterized, and therefore the statistical significance of a
matching genotype may be questionable. All these advantages of SNP
analysis are considerable in light of the consequences of most DNA
identification cases, which may lead to life imprisonment for an
individual, or re-association of remains to the family of a
deceased individual.
DNA can be isolated from biological samples such as blood, bone,
hair, saliva, or semen, and compared with the DNA from a reference
source at particular SNP positions. Multiple SNP markers can be
assayed simultaneously in order to increase the power of
discrimination and the statistical significance of a matching
genotype. For example, oligonucleotide arrays can be used to
genotype a large number of SNPs simultaneously. The SNPs provided
by the present invention can be assayed in combination with other
polymorphic genetic markers, such as other SNPs known in the art or
STRs, in order to identify an individual or to associate an
individual with a particular biological sample.
Furthermore, the SNPs provided by the present invention can be
genotyped for inclusion in a database of DNA genotypes, for
example, a criminal DNA databank such as the FBI's Combined DNA
Index System (CODIS) database. A genotype obtained from a
biological sample of unknown source can then be queried against the
database to find a matching genotype, with the SNPs of the present
invention providing nucleotide positions at which to compare the
known and unknown DNA sequences for identity. Accordingly, the
present invention provides a database comprising novel SNPs or SNP
alleles of the present invention (e.g., the database can comprise
information indicating which alleles are possessed by individual
members of a population at one or more novel SNP sites of the
present invention), such as for use in forensics, biometrics, or
other human identification applications. Such a database typically
comprises a computer-based system in which the SNPs or SNP alleles
of the present invention are recorded on a computer readable medium
(see the section of the present specification entitled
"Computer-Related Embodiments").
The SNPs of the present invention can also be assayed for use in
paternity testing. The object of paternity testing is usually to
determine whether a male is the father of a child. In most cases,
the mother of the child is known and thus, the mother's
contribution to the child's genotype can be traced. Paternity
testing investigates whether the part of the child's genotype not
attributable to the mother is consistent with that of the putative
father. Paternity testing can be performed by analyzing sets of
polymorphisms in the putative father and the child, with the SNPs
of the present invention providing nucleotide positions at which to
compare the putative father's and child's DNA sequences for
identity. If the set of polymorphisms in the child attributable to
the father does not match the set of polymorphisms of the putative
father, it can be concluded, barring experimental error, that the
putative father is not the father of the child. If the set of
polymorphisms in the child attributable to the father match the set
of polymorphisms of the putative father, a statistical calculation
can be performed to determine the probability of coincidental
match, and a conclusion drawn as to the likelihood that the
putative father is the true biological father of the child.
In addition to paternity testing, SNPs are also useful for other
types of kinship testing, such as for verifying familial
relationships for immigration purposes, or for cases in which an
individual alleges to be related to a deceased individual in order
to claim an inheritance from the deceased individual, etc. For
further information regarding the utility of SNPs for paternity
testing and other types of kinship testing, including methods for
statistical analysis, see Krawczak, "Informativity assessment for
biallelic single nucleotide polymorphisms", Electrophoresis 1999
June; 20(8):1676-81.
The use of the SNPs of the present invention for human
identification further extends to various authentication systems,
commonly referred to as biometric systems, which typically convert
physical characteristics of humans (or other organisms) into
digital data. Biometric systems include various technological
devices that measure such unique anatomical or physiological
characteristics as finger, thumb, or palm prints; hand geometry;
vein patterning on the back of the hand; blood vessel patterning of
the retina and color and texture of the iris; facial
characteristics; voice patterns; signature and typing dynamics; and
DNA. Such physiological measurements can be used to verify identity
and, for example, restrict or allow access based on the
identification. Examples of applications for biometrics include
physical area security, computer and network security, aircraft
passenger check-in and boarding, financial transactions, medical
records access, government benefit distribution, voting, law
enforcement, passports, visas and immigration, prisons, various
military applications, and for restricting access to expensive or
dangerous items, such as automobiles or guns (see, for example,
O'Connor, Stanford Technology Law Review and U.S. Pat. No.
6,119,096).
Groups of SNPs, particularly the SNPs provided by the present
invention, can be typed to uniquely identify an individual for
biometric applications such as those described above. Such SNP
typing can readily be accomplished using, for example, DNA
chips/arrays. Preferably, a minimally invasive means for obtaining
a DNA sample is utilized. For example, PCR amplification enables
sufficient quantities of DNA for analysis to be obtained from
buccal swabs or fingerprints, which contain DNA-containing skin
cells and oils that are naturally transferred during contact.
Further information regarding techniques for using SNPs in
forensic/human identification applications can be found in, for
example, Current Protocols in Human Genetics, John Wiley &
Sons, N.Y. (2002), 14.1-14.7.
Variant Proteins, Antibodies, Vectors & Host Cells, & Uses
Thereof
Variant Proteins Encoded by SNP-Containing Nucleic Acid
Molecules
The present invention provides SNP-containing nucleic acid
molecules, many of which encode proteins having variant amino acid
sequences as compared to the art-known (i.e., wild-type) proteins.
Amino acid sequences encoded by the polymorphic nucleic acid
molecules of the present invention are provided as SEQ ID
NOS:829-1656 in Table 1 and the Sequence Listing. These variants
will generally be referred to herein as variant
proteins/peptides/polypeptides, or polymorphic
proteins/peptides/polypeptides of the present invention. The terms
"protein", "peptide", and "polypeptide" are used herein
interchangeably.
A variant protein of the present invention may be encoded by, for
example, a nonsynonymous nucleotide substitution at any one of the
cSNP positions disclosed herein. In addition, variant proteins may
also include proteins whose expression, structure, and/or function
is altered by a SNP disclosed herein, such as a SNP that creates or
destroys a stop codon, a SNP that affects splicing, and a SNP in
control/regulatory elements, e.g. promoters, enhancers, or
transcription factor binding domains.
As used herein, a protein or peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
chemical precursors or other chemicals. The variant proteins of the
present invention can be purified to homogeneity or other lower
degrees of purity. The level of purification will be based on the
intended use. The key feature is that the preparation allows for
the desired function of the variant protein, even if in the
presence of considerable amounts of other components.
As used herein, "substantially free of cellular material" includes
preparations of the variant protein having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the variant protein is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
The language "substantially free of chemical precursors or other
chemicals" includes preparations of the variant protein in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the variant protein having less than about
30% (by dry weight) chemical precursors or other chemicals, less
than about 20% chemical precursors or other chemicals, less than
about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
An isolated variant protein may be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant host cells), or synthesized using known
protein synthesis methods. For example, a nucleic acid molecule
containing SNP(s) encoding the variant protein can be cloned into
an expression vector, the expression vector introduced into a host
cell, and the variant protein expressed in the host cell. The
variant protein can then be isolated from the cells by any
appropriate purification scheme using standard protein purification
techniques. Examples of these techniques are described in detail
below (Sambrook and Russell, 2000, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
The present invention provides isolated variant proteins that
comprise, consist of or consist essentially of amino acid sequences
that contain one or more variant amino acids encoded by one or more
codons which contain a SNP of the present invention.
Accordingly, the present invention provides variant proteins that
consist of amino acid sequences that contain one or more amino acid
polymorphisms (or truncations or extensions due to creation or
destruction of a stop codon, respectively) encoded by the SNPs
provided in Table 1 and/or Table 2. A protein consists of an amino
acid sequence when the amino acid sequence is the entire amino acid
sequence of the protein.
The present invention further provides variant proteins that
consist essentially of amino acid sequences that contain one or
more amino acid polymorphisms (or truncations or extensions due to
creation or destruction of a stop codon, respectively) encoded by
the SNPs provided in Table 1 and/or Table 2. A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues
in the final protein.
The present invention further provides variant proteins that
comprise amino acid sequences that contain one or more amino acid
polymorphisms (or truncations or extensions due to creation or
destruction of a stop codon, respectively) encoded by the SNPs
provided in Table 1 and/or Table 2. A protein comprises an amino
acid sequence when the amino acid sequence is at least part of the
final amino acid sequence of the protein. In such a fashion, the
protein may contain only the variant amino acid sequence or have
additional amino acid residues, such as a contiguous encoded
sequence that is naturally associated with it or heterologous amino
acid residues. Such a protein can have a few additional amino acid
residues or can comprise many more additional amino acids. A brief
description of how various types of these proteins can be made and
isolated is provided below.
The variant proteins of the present invention can be attached to
heterologous sequences to form chimeric or fusion proteins. Such
chimeric and fusion proteins comprise a variant protein operatively
linked to a heterologous protein having an amino acid sequence not
substantially homologous to the variant protein. "Operatively
linked" indicates that the coding sequences for the variant protein
and the heterologous protein are ligated in-frame. The heterologous
protein can be fused to the N-terminus or C-terminus of the variant
protein. In another embodiment, the fusion protein is encoded by a
fusion polynucleotide that is synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
re-amplified to generate a chimeric gene sequence (see Ausubel et
al., Current Protocols in Molecular Biology, 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST protein). A variant protein-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the variant protein.
In many uses, the fusion protein does not affect the activity of
the variant protein. The fusion protein can include, but is not
limited to, enzymatic fusion proteins, for example,
beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His
fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate their
purification following recombinant expression. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Fusion proteins are further described in, for example, Terpe,
"Overview of tag protein fusions: from molecular and biochemical
fundamentals to commercial systems", Appl Microbiol Biotechnol.
2003 January; 60(5):523-33. Epub 2002 Nov. 07; Graddis et al.,
"Designing proteins that work using recombinant technologies", Curr
Pharm Biotechnol. 2002 December; 3(4):285-97; and Nilsson et al.,
"Affinity fusion strategies for detection, purification, and
immobilization of recombinant proteins", Protein Expr Purif. 1997
October; 11(1):1-16.
The present invention also relates to further obvious variants of
the variant polypeptides of the present invention, such as
naturally-occurring mature forms (e.g., alleleic variants),
non-naturally occurring recombinantly-derived variants, and
orthologs and paralogs of such proteins that share sequence
homology. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry. It is understood, however, that variants
exclude those known in the prior art before the present
invention.
Further variants of the variant polypeptides disclosed in Table 1
can comprise an amino acid sequence that shares at least 70-80%,
80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity with an amino acid sequence disclosed in Table 1
(or a fragment thereof) and that includes a novel amino acid
residue (allele) disclosed in Table 1 (which is encoded by a novel
SNP allele). Thus, the present invention specifically contemplates
polypeptides that have a certain degree of sequence variation
compared with the polypeptide sequences shown in Table 1, but that
contain a novel amino acid residue (allele) encoded by a novel SNP
allele disclosed herein. In other words, as long as a polypeptide
contains a novel amino acid residue disclosed herein, other
portions of the polypeptide that flank the novel amino acid residue
can vary to some degree from the polypeptide sequences shown in
Table 1.
Full-length pre-processed forms, as well as mature processed forms,
of proteins that comprise one of the amino acid sequences disclosed
herein can readily be identified as having complete sequence
identity to one of the variant proteins of the present invention as
well as being encoded by the same genetic locus as the variant
proteins provided herein.
Orthologs of a variant peptide can readily be identified as having
some degree of significant sequence homology/identity to at least a
portion of a variant peptide as well as being encoded by a gene
from another organism. Preferred orthologs will be isolated from
non-human mammals, preferably primates, for the development of
human therapeutic targets and agents. Such orthologs can be encoded
by a nucleic acid sequence that hybridizes to a variant
peptide-encoding nucleic acid molecule under moderate to stringent
conditions depending on the degree of relatedness of the two
organisms yielding the homologous proteins.
Variant proteins include, but are not limited to, proteins
containing deletions, additions and substitutions in the amino acid
sequence caused by the SNPs of the present invention. One class of
substitutions is conserved amino acid substitutions in which a
given amino acid in a polypeptide is substituted for another amino
acid of like characteristics. Typical conservative substitutions
are replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution
between the amide residues Asn and Gln; exchange of the basic
residues Lys and Arg; and replacements among the aromatic residues
Phe and Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in, for example, Bowie
et al., Science 247:1306-1310 (1990).
Variant proteins can be fully functional or can lack function in
one or more activities, e.g. ability to bind another molecule,
ability to catalyze a substrate, ability to mediate signaling, etc.
Fully functional variants typically contain only conservative
variations or variations in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids that result in no change or an
insignificant change in function. Alternatively, such substitutions
may positively or negatively affect function to some degree.
Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, truncations or extensions, or a substitution,
insertion, inversion, or deletion of a critical residue or in a
critical region.
Amino acids that are essential for function of a protein can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the amino acid
sequence and polymorphism information provided in Table 1. The
latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as enzyme activity or in assays
such as an in vitro proliferative activity. Sites that are critical
for binding partner/substrate binding can also be determined by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al., J. Mol. Biol.
224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).
Polypeptides can contain amino acids other than the 20 amino acids
commonly referred to as the 20 naturally occurring amino acids.
Further, many amino acids, including the terminal amino acids, may
be modified by natural processes, such as processing and other
post-translational modifications, or by chemical modification
techniques well known in the art. Accordingly, the variant proteins
of the present invention also encompass derivatives or analogs in
which a substituted amino acid residue is not one encoded by the
genetic code, in which a substituent group is included, in which
the mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (e.g.,
polyethylene glycol), or in which additional amino acids are fused
to the mature polypeptide, such as a leader or secretory sequence
or a sequence for purification of the mature polypeptide or a
pro-protein sequence.
Known protein modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
Such protein modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993); Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al., Meth.
Enzymol. 182: 626-646 (1990); and Rattan et al., Ann. N Y Acad.
Sci. 663:48-62 (1992).
The present invention further provides fragments of the variant
proteins in which the fragments contain one or more amino acid
sequence variations (e.g., substitutions, or truncations or
extensions due to creation or destruction of a stop codon) encoded
by one or more SNPs disclosed herein. The fragments to which the
invention pertains, however, are not to be construed as
encompassing fragments that have been disclosed in the prior art
before the present invention.
As used herein, a fragment may comprise at least about 4, 8, 10,
12, 14, 16, 18, 20, 25, 30, 50, 100 (or any other number
in-between) or more contiguous amino acid residues from a variant
protein, wherein at least one amino acid residue is affected by a
SNP of the present invention, e.g., a variant amino acid residue
encoded by a nonsynonymous nucleotide substitution at a cSNP
position provided by the present invention. The variant amino acid
encoded by a cSNP may occupy any residue position along the
sequence of the fragment. Such fragments can be chosen based on the
ability to retain one or more of the biological activities of the
variant protein or the ability to perform a function, e.g., act as
an immunogen. Particularly important fragments are biologically
active fragments. Such fragments will typically comprise a domain
or motif of a variant protein of the present invention, e.g.,
active site, transmembrane domain, or ligand/substrate binding
domain. Other fragments include, but are not limited to, domain or
motif-containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known to those of skill in the art (e.g., PROSITE analysis)
(Current Protocols in Protein Science, John Wiley & Sons, N.Y.
(2002)).
Uses of Variant Proteins
The variant proteins of the present invention can be used in a
variety of ways, including but not limited to, in assays to
determine the biological activity of a variant protein, such as in
a panel of multiple proteins for high-throughput screening; to
raise antibodies or to elicit another type of immune response; as a
reagent (including the labeled reagent) in assays designed to
quantitatively determine levels of the variant protein (or its
binding partner) in biological fluids; as a marker for cells or
tissues in which it is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); as a target for screening
for a therapeutic agent; and as a direct therapeutic agent to be
administered into a human subject. Any of the variant proteins
disclosed herein may be developed into reagent grade or kit format
for commercialization as research products. Methods for performing
the uses listed above are well known to those skilled in the art
(see, e.g., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Sambrook and Russell, 2000, and Methods in
Enzymology: Guide to Molecular Cloning Techniques, Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987).
In a specific embodiment of the invention, the methods of the
present invention include detection of one or more variant proteins
disclosed herein. Variant proteins are disclosed in Table 1 and in
the Sequence Listing as SEQ ID NOS: 829-1656. Detection of such
proteins can be accomplished using, for example, antibodies, small
molecule compounds, aptamers, ligands/substrates, other proteins or
protein fragments, or other protein-binding agents. Preferably,
protein detection agents are specific for a variant protein of the
present invention and can therefore discriminate between a variant
protein of the present invention and the wild-type protein or
another variant form. This can generally be accomplished by, for
example, selecting or designing detection agents that bind to the
region of a protein that differs between the variant and wild-type
protein, such as a region of a protein that contains one or more
amino acid substitutions that is/are encoded by a non-synonymous
cSNP of the present invention, or a region of a protein that
follows a nonsense mutation-type SNP that creates a stop codon
thereby leading to a shorter polypeptide, or a region of a protein
that follows a read-through mutation-type SNP that destroys a stop
codon thereby leading to a longer polypeptide in which a portion of
the polypeptide is present in one version of the polypeptide but
not the other.
In another specific aspect of the invention, the variant proteins
of the present invention are used as targets for diagnosing
myocardial infarction or for determining predisposition to
myocardial infarction in a human. Accordingly, the invention
provides methods for detecting the presence of, or levels of, one
or more variant proteins of the present invention in a cell,
tissue, or organism. Such methods typically involve contacting a
test sample with an agent (e.g., an antibody, small molecule
compound, or peptide) capable of interacting with the variant
protein such that specific binding of the agent to the variant
protein can be detected. Such an assay can be provided in a single
detection format or a multi-detection format such as an array, for
example, an antibody or aptamer array (arrays for protein detection
may also be referred to as "protein chips"). The variant protein of
interest can be isolated from a test sample and assayed for the
presence of a variant amino acid sequence encoded by one or more
SNPs disclosed by the present invention. The SNPs may cause changes
to the protein and the corresponding protein function/activity,
such as through non-synonymous substitutions in protein coding
regions that can lead to amino acid substitutions, deletions,
insertions, and/or rearrangements; formation or destruction of stop
codons; or alteration of control elements such as promoters. SNPs
may also cause inappropriate post-translational modifications.
One preferred agent for detecting a variant protein in a sample is
an antibody capable of selectively binding to a variant form of the
protein (antibodies are described in greater detail in the next
section). Such samples include, for example, tissues, cells, and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject.
In vitro methods for detection of the variant proteins associated
with myocardial infarction that are disclosed herein and fragments
thereof include, but are not limited to, enzyme linked
immunosorbent assays (ELISAs), radioimmunoassays (RIA), Western
blots, immunoprecipitations, immunofluorescence, and protein
arrays/chips (e.g., arrays of antibodies or aptamers). For further
information regarding immunoassays and related protein detection
methods, see Current Protocols in Immunology, John Wiley &
Sons, N.Y., and Hage, "Immunoassays", Anal Chem. 1999 Jun. 15;
71(12):294R-304R.
Additional analytic methods of detecting amino acid variants
include, but are not limited to, altered electrophoretic mobility,
altered tryptic peptide digest, altered protein activity in
cell-based or cell-free assay, alteration in ligand or
antibody-binding pattern, altered isoelectric point, and direct
amino acid sequencing.
Alternatively, variant proteins can be detected in vivo in a
subject by introducing into the subject a labeled antibody (or
other type of detection reagent) specific for a variant protein.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
Other uses of the variant peptides of the present invention are
based on the class or action of the protein. For example, proteins
isolated from humans and their mammalian orthologs serve as targets
for identifying agents (e.g., small molecule drugs or antibodies)
for use in therapeutic applications, particularly for modulating a
biological or pathological response in a cell or tissue that
expresses the protein. Pharmaceutical agents can be developed that
modulate protein activity.
As an alternative to modulating gene expression, therapeutic
compounds can be developed that modulate protein function. For
example, many SNPs disclosed herein affect the amino acid sequence
of the encoded protein (e.g., non-synonymous cSNPs and nonsense
mutation-type SNPs). Such alterations in the encoded amino acid
sequence may affect protein function, particularly if such amino
acid sequence variations occur in functional protein domains, such
as catalytic domains, ATP-binding domains, or ligand/substrate
binding domains. It is well established in the art that variant
proteins having amino acid sequence variations in functional
domains can cause or influence pathological conditions. In such
instances, compounds (e.g., small molecule drugs or antibodies) can
be developed that target the variant protein and modulate (e.g.,
up- or down-regulate) protein function/activity.
The therapeutic methods of the present invention further include
methods that target one or more variant proteins of the present
invention. Variant proteins can be targeted using, for example,
small molecule compounds, antibodies, aptamers, ligands/substrates,
other proteins, or other protein-binding agents. Additionally, the
skilled artisan will recognize that the novel protein variants (and
polymorphic nucleic acid molecules) disclosed in Table 1 may
themselves be directly used as therapeutic agents by acting as
competitive inhibitors of corresponding art-known proteins (or
nucleic acid molecules such as mRNA molecules).
The variant proteins of the present invention are particularly
useful in drug screening assays, in cell-based or cell-free
systems. Cell-based systems can utilize cells that naturally
express the protein, a biopsy specimen, or cell cultures. In one
embodiment, cell-based assays involve recombinant host cells
expressing the variant protein. Cell-free assays can be used to
detect the ability of a compound to directly bind to a variant
protein or to the corresponding SNP-containing nucleic acid
fragment that encodes the variant protein.
A variant protein of the present invention, as well as appropriate
fragments thereof, can be used in high-throughput screening assays
to test candidate compounds for the ability to bind and/or modulate
the activity of the variant protein. These candidate compounds can
be further screened against a protein having normal function (e.g.,
a wild-type/non-variant protein) to further determine the effect of
the compound on the protein activity. Furthermore, these compounds
can be tested in animal or invertebrate systems to determine in
vivo activity/effectiveness. Compounds can be identified that
activate (agonists) or inactivate (antagonists) the variant
protein, and different compounds can be identified that cause
various degrees of activation or inactivation of the variant
protein.
Further, the variant proteins can be used to screen a compound for
the ability to stimulate or inhibit interaction between the variant
protein and a target molecule that normally interacts with the
protein. The target can be a ligand, a substrate or a binding
partner that the protein normally interacts with (for example,
epinephrine or norepinephrine). Such assays typically include the
steps of combining the variant protein with a candidate compound
under conditions that allow the variant protein, or fragment
thereof, to interact with the target molecule, and to detect the
formation of a complex between the protein and the target or to
detect the biochemical consequence of the interaction with the
variant protein and the target, such as any of the associated
effects of signal transduction.
Candidate compounds include, for example, 1) peptides such as
soluble peptides, including Ig-tailed fusion peptides and members
of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L- configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
One candidate compound is a soluble fragment of the variant protein
that competes for ligand binding. Other candidate compounds include
mutant proteins or appropriate fragments containing mutations that
affect variant protein function and thus compete for ligand.
Accordingly, a fragment that competes for ligand, for example with
a higher affinity, or a fragment that binds ligand but does not
allow release, is encompassed by the invention.
The invention further includes other end point assays to identify
compounds that modulate (stimulate or inhibit) variant protein
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate protein activity. Thus,
the expression of genes that are up or down-regulated in response
to the variant protein dependent signal cascade can be assayed. In
one embodiment, the regulatory region of such genes can be operably
linked to a marker that is easily detectable, such as luciferase.
Alternatively, phosphorylation of the variant protein, or a variant
protein target, could also be measured. Any of the biological or
biochemical functions mediated by the variant protein can be used
as an endpoint assay. These include all of the biochemical or
biological events described herein, in the references cited herein,
incorporated by reference for these endpoint assay targets, and
other functions known to those of ordinary skill in the art.
Binding and/or activating compounds can also be screened by using
chimeric variant proteins in which an amino terminal extracellular
domain or parts thereof, an entire transmembrane domain or
subregions, and/or the carboxyl terminal intracellular domain or
parts thereof, can be replaced by heterologous domains or
subregions. For example, a substrate-binding region can be used
that interacts with a different substrate than that which is
normally recognized by a variant protein. Accordingly, a different
set of signal transduction components is available as an end-point
assay for activation. This allows for assays to be performed in
other than the specific host cell from which the variant protein is
derived.
The variant proteins are also useful in competition binding assays
in methods designed to discover compounds that interact with the
variant protein. Thus, a compound can be exposed to a variant
protein under conditions that allow the compound to bind or to
otherwise interact with the variant protein. A binding partner,
such as ligand, that normally interacts with the variant protein is
also added to the mixture. If the test compound interacts with the
variant protein or its binding partner, it decreases the amount of
complex formed or activity from the variant protein. This type of
assay is particularly useful in screening for compounds that
interact with specific regions of the variant protein (Hodgson,
Bio/technology, 1992, September 10(9), 973-80).
To perform cell-free drug screening assays, it is sometimes
desirable to immobilize either the variant protein or a fragment
thereof, or its target molecule, to facilitate separation of
complexes from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Any method for
immobilizing proteins on matrices can be used in drug screening
assays. In one embodiment, a fusion protein containing an added
domain allows the protein to be bound to a matrix. For example,
glutathione-S-transferase/.sup.125I fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the cell lysates (e.g., .sup.35S-labeled) and a
candidate compound, such as a drug candidate, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads can be washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of bound material found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques.
Either the variant protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Alternatively, antibodies reactive with the variant protein but
which do not interfere with binding of the variant protein to its
target molecule can be derivatized to the wells of the plate, and
the variant protein trapped in the wells by antibody conjugation.
Preparations of the target molecule and a candidate compound are
incubated in the variant protein-presenting wells and the amount of
complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the protein target molecule, or
which are reactive with variant protein and compete with the target
molecule, and enzyme-linked assays that rely on detecting an
enzymatic activity associated with the target molecule.
Modulators of variant protein activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the protein pathway, such as myocardial
infarction. These methods of treatment typically include the steps
of administering the modulators of protein activity in a
pharmaceutical composition to a subject in need of such
treatment.
The variant proteins, or fragments thereof, disclosed herein can
themselves be directly used to treat a disorder characterized by an
absence of, inappropriate, or unwanted expression or activity of
the variant protein. Accordingly, methods for treatment include the
use of a variant protein disclosed herein or fragments thereof.
In yet another aspect of the invention, variant proteins can be
used as "bait proteins" in a two-hybrid assay or three-hybrid assay
(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1693-1696; and Brent WO94/10300) to identify
other proteins that bind to or interact with the variant protein
and are involved in variant protein activity. Such variant
protein-binding proteins are also likely to be involved in the
propagation of signals by the variant proteins or variant protein
targets as, for example, elements of a protein-mediated signaling
pathway. Alternatively, such variant protein-binding proteins are
inhibitors of the variant protein.
The two-hybrid system is based on the modular nature of most
transcription factors, which typically consist of separable
DNA-binding and activation domains. Briefly, the assay typically
utilizes two different DNA constructs. In one construct, the gene
that codes for a variant protein is fused to a gene encoding the
DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or
"sample") is fused to a gene that codes for the activation domain
of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming a variant
protein-dependent complex, the DNA-binding and activation domains
of the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ) that
is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be
detected, and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene that
encodes the protein that interacts with the variant protein.
Antibodies Directed to Variant Proteins
The present invention also provides antibodies that selectively
bind to the variant proteins disclosed herein and fragments
thereof. Such antibodies may be used to quantitatively or
qualitatively detect the variant proteins of the present invention.
As used herein, an antibody selectively binds a target variant
protein when it binds the variant protein and does not
significantly bind to non-variant proteins, i.e., the antibody does
not significantly bind to normal, wild-type, or art-known proteins
that do not contain a variant amino acid sequence due to one or
more SNPs of the present invention (variant amino acid sequences
may be due to, for example, nonsynonymous cSNPs, nonsense SNPs that
create a stop codon, thereby causing a truncation of a polypeptide
or SNPs that cause read-through mutations resulting in an extension
of a polypeptide).
As used herein, an antibody is defined in terms consistent with
that recognized in the art: they are multi-subunit proteins
produced by an organism in response to an antigen challenge. The
antibodies of the present invention include both monoclonal
antibodies and polyclonal antibodies, as well as antigen-reactive
proteolytic fragments of such antibodies, such as Fab,
F(ab)'.sub.2, and Fv fragments. In addition, an antibody of the
present invention further includes any of a variety of engineered
antigen-binding molecules such as a chimeric antibody (U.S. Pat.
Nos. 4,816,567 and 4,816,397; Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851, 1984; Neuberger et al., Nature 312:604, 1984), a
humanized antibody (U.S. Pat. Nos. 5,693,762; 5,585,089; and
5,565,332), a single-chain Fv (U.S. Pat. No. 4,946,778; Ward et
al., Nature 334:544, 1989), a bispecific antibody with two binding
specificities (Segal et al., J. Immunol. Methods 248:1, 2001;
Carter, J. Immunol. Methods 248:7, 2001), a diabody, a triabody,
and a tetrabody (Todorovska et al., J. Immunol. Methods, 248:47,
2001), as well as a Fab conjugate (dimer or trimer), and a
minibody.
Many methods are known in the art for generating and/or identifying
antibodies to a given target antigen (Harlow, Antibodies, Cold
Spring Harbor Press, (1989)). In general, an isolated peptide
(e.g., a variant protein of the present invention) is used as an
immunogen and is administered to a mammalian organism, such as a
rat, rabbit, hamster or mouse. Either a full-length protein, an
antigenic peptide fragment (e.g., a peptide fragment containing a
region that varies between a variant protein and a corresponding
wild-type protein), or a fusion protein can be used. A protein used
as an immunogen may be naturally-occurring, synthetic or
recombinantly produced, and may be administered in combination with
an adjuvant, including but not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substance such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and the like.
Monoclonal antibodies can be produced by hybridoma technology
(Kohler and Milstein, Nature, 256:495, 1975), which immortalizes
cells secreting a specific monoclonal antibody. The immortalized
cell lines can be created in vitro by fusing two different cell
types, typically lymphocytes, and tumor cells. The hybridoma cells
may be cultivated in vitro or in vivo. Additionally, fully human
antibodies can be generated by transgenic animals (He et al., J.
Immunol., 169:595, 2002). Fd phage and Fd phagemid technologies may
be used to generate and select recombinant antibodies in vitro
(Hoogenboom and Chames, Immunol. Today 21:371, 2000; Liu et al., J.
Mol. Biol. 315:1063, 2002). The complementarity-determining regions
of an antibody can be identified, and synthetic peptides
corresponding to such regions may be used to mediate antigen
binding (U.S. Pat. No. 5,637,677).
Antibodies are preferably prepared against regions or discrete
fragments of a variant protein containing a variant amino acid
sequence as compared to the corresponding wild-type protein (e.g.,
a region of a variant protein that includes an amino acid encoded
by a nonsynonymous cSNP, a region affected by truncation caused by
a nonsense SNP that creates a stop codon, or a region resulting
from the destruction of a stop codon due to read-through mutation
caused by a SNP). Furthermore, preferred regions will include those
involved in function/activity and/or protein/binding partner
interaction. Such fragments can be selected on a physical property,
such as fragments corresponding to regions that are located on the
surface of the protein, e.g., hydrophilic regions, or can be
selected based on sequence uniqueness, or based on the position of
the variant amino acid residue(s) encoded by the SNPs provided by
the present invention. An antigenic fragment will typically
comprise at least about 8-10 contiguous amino acid residues in
which at least one of the amino acid residues is an amino acid
affected by a SNP disclosed herein. The antigenic peptide can
comprise, however, at least 12, 14, 16, 20, 25, 50, 100 (or any
other number in-between) or more amino acid residues, provided that
at least one amino acid is affected by a SNP disclosed herein.
Detection of an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody or
an antigen-reactive fragment thereof to a detectable substance.
Detectable substances include, but are not limited to, various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, O-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
Antibodies, particularly the use of antibodies as therapeutic
agents, are reviewed in: Morgan, "Antibody therapy for Alzheimer's
disease", Expert Rev Vaccines. 2003 February; 2(1):53-9; Ross et
al., "Anticancer antibodies", Am J Clin Pathol. 2003 April;
119(4):472-85; Goldenberg, "Advancing role of radiolabeled
antibodies in the therapy of cancer", Cancer Immunol Immunother.
2003 May; 52(5):281-96. Epub 2003 March 11; Ross et al.,
"Antibody-based therapeutics in oncology", Expert Rev Anticancer
Ther. 2003 February; 3(1):107-21; Cao et al., "Bispecific antibody
conjugates in therapeutics", Adv Drug Deliv Rev. 2003 February 10;
55(2):171-97; von Mehren et al., "Monoclonal antibody therapy for
cancer", Annu Rev Med. 2003; 54:343-69. Epub 2001 December 03;
Hudson et al., "Engineered antibodies", Nat Med. 2003 January;
9(1):129-34; Brekke et al., "Therapeutic antibodies for human
diseases at the dawn of the twenty-first century", Nat Rev Drug
Discov. 2003 January; 2(1):52-62 (Erratum in: Nat Rev Drug Discov.
2003 March; 2(3):240); Houdebine, "Antibody manufacture in
transgenic animals and comparisons with other systems", Curr Opin
Biotechnol. 2002 December; 13(6):625-9; Andreakos et al.,
"Monoclonal antibodies in immune and inflammatory diseases", Curr
Opin Biotechnol. 2002 December; 13(6):615-20; Kellermann et al.,
"Antibody discovery: the use of transgenic mice to generate human
monoclonal antibodies for therapeutics", Curr Opin Biotechnol. 2002
December; 13(6):593-7; Pini et al., "Phage display and colony
filter screening for high-throughput selection of antibody
libraries", Comb Chem High Throughput Screen. 2002 November;
5(7):503-10; Batra et al., "Pharmacokinetics and biodistribution of
genetically engineered antibodies", Curr Opin Biotechnol. 2002
December; 13(6):603-8; and Tangri et al., "Rationally engineered
proteins or antibodies with absent or reduced immunogenicity", Curr
Med Chem. 2002 December; 9(24):2191-9.
Uses of Antibodies
Antibodies can be used to isolate the variant proteins of the
present invention from a natural cell source or from recombinant
host cells by standard techniques, such as affinity chromatography
or immunoprecipitation. In addition, antibodies are useful for
detecting the presence of a variant protein of the present
invention in cells or tissues to determine the pattern of
expression of the variant protein among various tissues in an
organism and over the course of normal development or disease
progression. Further, antibodies can be used to detect variant
protein in situ, in vitro, in a bodily fluid, or in a cell lysate
or supernatant in order to evaluate the amount and pattern of
expression. Also, antibodies can be used to assess abnormal tissue
distribution, abnormal expression during development, or expression
in an abnormal condition, such as myocardial infarction.
Additionally, antibody detection of circulating fragments of the
full-length variant protein can be used to identify turnover.
Antibodies to the variant proteins of the present invention are
also useful in pharmacogenomic analysis. Thus, antibodies against
variant proteins encoded by alternative SNP alleles can be used to
identify individuals that require modified treatment
modalities.
Further, antibodies can be used to assess expression of the variant
protein in disease states such as in active stages of the disease
or in an individual with a predisposition to a disease related to
the protein's function, particularly myocardial infarction.
Antibodies specific for a variant protein encoded by a
SNP-containing nucleic acid molecule of the present invention can
be used to assay for the presence of the variant protein, such as
to screen for predisposition to myocardial infarction as indicated
by the presence of the variant protein.
Antibodies are also useful as diagnostic tools for evaluating the
variant proteins in conjunction with analysis by electrophoretic
mobility, isoelectric point, tryptic peptide digest, and other
physical assays well known in the art.
Antibodies are also useful for tissue typing. Thus, where a
specific variant protein has been correlated with expression in a
specific tissue, antibodies that are specific for this protein can
be used to identify a tissue type.
Antibodies can also be used to assess aberrant subcellular
localization of a variant protein in cells in various tissues. The
diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting the expression level or
the presence of variant protein or aberrant tissue distribution or
developmental expression of a variant protein, antibodies directed
against the variant protein or relevant fragments can be used to
monitor therapeutic efficacy.
The antibodies are also useful for inhibiting variant protein
function, for example, by blocking the binding of a variant protein
to a binding partner. These uses can also be applied in a
therapeutic context in which treatment involves inhibiting a
variant protein's function. An antibody can be used, for example,
to block or competitively inhibit binding, thus modulating
(agonizing or antagonizing) the activity of a variant protein.
Antibodies can be prepared against specific variant protein
fragments containing sites required for function or against an
intact variant protein that is associated with a cell or cell
membrane. For in vivo administration, an antibody may be linked
with an additional therapeutic payload such as a radionuclide, an
enzyme, an immunogenic epitope, or a cytotoxic agent. Suitable
cytotoxic agents include, but are not limited to, bacterial toxin
such as diphtheria, and plant toxin such as ricin. The in vivo
half-life of an antibody or a fragment thereof may be lengthened by
pegylation through conjugation to polyethylene glycol (Leong et
al., Cytokine 16:106, 2001).
The invention also encompasses kits for using antibodies, such as
kits for detecting the presence of a variant protein in a test
sample. An exemplary kit can comprise antibodies such as a labeled
or labelable antibody and a compound or agent for detecting variant
proteins in a biological sample; means for determining the amount,
or presence/absence of variant protein in the sample; means for
comparing the amount of variant protein in the sample with a
standard; and instructions for use.
Vectors and Host Cells
The present invention also provides vectors containing the
SNP-containing nucleic acid molecules described herein. The term
"vector" refers to a vehicle, preferably a nucleic acid molecule,
which can transport a SNP-containing nucleic acid molecule. When
the vector is a nucleic acid molecule, the SNP-containing nucleic
acid molecule can be covalently linked to the vector nucleic acid.
Such vectors include, but are not limited to, a plasmid, single or
double stranded phage, a single or double stranded RNA or DNA viral
vector, or artificial chromosome, such as a BAC, PAC, YAC, or
MAC.
A vector can be maintained in a host cell as an extrachromosomal
element where it replicates and produces additional copies of the
SNP-containing nucleic acid molecules. Alternatively, the vector
may integrate into the host cell genome and produce additional
copies of the SNP-containing nucleic acid molecules when the host
cell replicates.
The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
SNP-containing nucleic acid molecules. The vectors can function in
prokaryotic or eukaryotic cells or in both (shuttle vectors).
Expression vectors typically contain cis-acting regulatory regions
that are operably linked in the vector to the SNP-containing
nucleic acid molecules such that transcription of the
SNP-containing nucleic acid molecules is allowed in a host cell.
The SNP-containing nucleic acid molecules can also be introduced
into the host cell with a separate nucleic acid molecule capable of
affecting transcription. Thus, the second nucleic acid molecule may
provide a trans-acting factor interacting with the cis-regulatory
control region to allow transcription of the SNP-containing nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
The regulatory sequences to which the SNP-containing nucleic acid
molecules described herein can be operably linked include promoters
for directing mRNA transcription. These include, but are not
limited to, the left promoter from bacteriophage .lamda., the lac,
TRP, and TAC promoters from E. coli, the early and late promoters
from SV40, the CMV immediate early promoter, the adenovirus early
and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
In addition to containing sites for transcription initiation and
control, expression vectors can also contain sequences necessary
for transcription termination and, in the transcribed region, a
ribosome-binding site for translation. Other regulatory control
elements for expression include initiation and termination codons
as well as polyadenylation signals. A person of ordinary skill in
the art would be aware of the numerous regulatory sequences that
are useful in expression vectors (see, e.g., Sambrook and Russell,
2000, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
A variety of expression vectors can be used to express a
SNP-containing nucleic acid molecule. Such vectors include
chromosomal, episomal, and virus-derived vectors, for example,
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, including yeast
artificial chromosomes, from viruses such as baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors can
also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, e.g.,
cosmids and phagemids. Appropriate cloning and expression vectors
for prokaryotic and eukaryotic hosts are described in Sambrook and
Russell, 2000, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
The regulatory sequence in a vector may provide constitutive
expression in one or more host cells (e.g., tissue specific
expression) or may provide for inducible expression in one or more
cell types such as by temperature, nutrient additive, or exogenous
factor, e.g., a hormone or other ligand. A variety of vectors that
provide constitutive or inducible expression of a nucleic acid
sequence in prokaryotic and eukaryotic host cells are well known to
those of ordinary skill in the art.
A SNP-containing nucleic acid molecule can be inserted into the
vector by methodology well-known in the art. Generally, the
SNP-containing nucleic acid molecule that will ultimately be
expressed is joined to an expression vector by cleaving the
SNP-containing nucleic acid molecule and the expression vector with
one or more restriction enzymes and then ligating the fragments
together. Procedures for restriction enzyme digestion and ligation
are well known to those of ordinary skill in the art.
The vector containing the appropriate nucleic acid molecule can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques.
Bacterial host cells include, but are not limited to, E. coli,
Streptomyces, and Salmonella typhimurium. Eukaryotic host cells
include, but are not limited to, yeast, insect cells such as
Drosophila, animal cells such as COS and CHO cells, and plant
cells.
As described herein, it may be desirable to express the variant
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the variant
peptides. Fusion vectors can, for example, increase the expression
of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting, for example, as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired variant peptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes suitable for
such use include, but are not limited to, factor Xa, thrombin, and
enterokinase. Typical fusion expression vectors include pGEX (Smith
et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly,
Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185:60-89 (1990)).
Recombinant protein expression can be maximized in a bacterial host
by providing a genetic background wherein the host cell has an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128).
Alternatively, the sequence of the SNP-containing nucleic acid
molecule of interest can be altered to provide preferential codon
usage for a specific host cell, for example, E. coli (Wada et al.,
Nucleic Acids Res. 20:2111-2118 (1992)).
The SNP-containing nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast (e.g., S. cerevisiae) include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
The SNP-containing nucleic acid molecules can also be expressed in
insect cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
In certain embodiments of the invention, the SNP-containing nucleic
acid molecules described herein are expressed in mammalian cells
using mammalian expression vectors. Examples of mammalian
expression vectors include pCDM8 (Seed, B. Nature 329:840(1987))
and pMT2PC (Kaufinan et al., EMBO J. 6:187-195 (1987)).
The invention also encompasses vectors in which the SNP-containing
nucleic acid molecules described herein are cloned into the vector
in reverse orientation, but operably linked to a regulatory
sequence that permits transcription of antisense RNA. Thus, an
antisense transcript can be produced to the SNP-containing nucleic
acid sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the
vectors described herein. Host cells therefore include, for
example, prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
The recombinant host cells can be prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to persons of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those described in
Sambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.).
Host cells can contain more than one vector. Thus, different
SNP-containing nucleotide sequences can be introduced in different
vectors into the same cell. Similarly, the SNP-containing nucleic
acid molecules can be introduced either alone or with other nucleic
acid molecules that are not related to the SNP-containing nucleic
acid molecules, such as those providing trans-acting factors for
expression vectors. When more than one vector is introduced into a
cell, the vectors can be introduced independently, co-introduced,
or joined to the nucleic acid molecule vector.
In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication can occur in host
cells that provide functions that complement the defects.
Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be inserted in the
same vector that contains the SNP-containing nucleic acid molecules
described herein or may be in a separate vector. Markers include,
for example, tetracycline or ampicillin-resistance genes for
prokaryotic host cells, and dihydrofolate reductase or neomycin
resistance genes for eukaryotic host cells. However, any marker
that provides selection for a phenotypic trait can be
effective.
While the mature variant proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these variant
proteins using RNA derived from the DNA constructs described
herein.
Where secretion of the variant protein is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as G-protein-coupled receptors (GPCRs), appropriate
secretion signals can be incorporated into the vector. The signal
sequence can be endogenous to the peptides or heterologous to these
peptides.
Where the variant protein is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze/thaw, sonication, mechanical
disruption, use of lysing agents, and the like. The variant protein
can then be recovered and purified by well-known purification
methods including, for example, ammonium sulfate precipitation,
acid extraction, anion or cationic exchange chromatography,
phosphocellulose chromatography, hydrophobic-interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, lectin chromatography, or high performance liquid
chromatography.
It is also understood that, depending upon the host cell in which
recombinant production of the variant proteins described herein
occurs, they can have various glycosylation patterns, or may be
non-glycosylated, as when produced in bacteria. In addition, the
variant proteins may include an initial modified methionine in some
cases as a result of a host-mediated process.
For further information regarding vectors and host cells, see
Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y.
Uses of Vectors and Host Cells, and Transgenic Animals
Recombinant host cells that express the variant proteins described
herein have a variety of uses. For example, the cells are useful
for producing a variant protein that can be further purified into a
preparation of desired amounts of the variant protein or fragments
thereof. Thus, host cells containing expression vectors are useful
for variant protein production.
Host cells are also useful for conducting cell-based assays
involving the variant protein or variant protein fragments, such as
those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a variant protein is
useful for assaying compounds that stimulate or inhibit variant
protein function. Such an ability of a compound to modulate variant
protein function may not be apparent from assays of the compound on
the native/wild-type protein, or from cell-free assays of the
compound. Recombinant host cells are also useful for assaying
functional alterations in the variant proteins as compared with a
known function.
Genetically-engineered host cells can be further used to produce
non-human transgenic animals. A transgenic animal is preferably a
non-human mammal, for example, a rodent, such as a rat or mouse, in
which one or more of the cells of the animal include a transgene. A
transgene is exogenous DNA containing a SNP of the present
invention which is integrated into the genome of a cell from which
a transgenic animal develops and which remains in the genome of the
mature animal in one or more of its cell types or tissues. Such
animals are useful for studying the function of a variant protein
in vivo, and identifying and evaluating modulators of variant
protein activity. Other examples of transgenic animals include, but
are not limited to, non-human primates, sheep, dogs, cows, goats,
chickens, and amphibians. Transgenic non-human mammals such as cows
and goats can be used to produce variant proteins which can be
secreted in the animal's milk and then recovered.
A transgenic animal can be produced by introducing a SNP-containing
nucleic acid molecule into the male pronuclei of a fertilized
oocyte, e.g., by microinjection or retroviral infection, and
allowing the oocyte to develop in a pseudopregnant female foster
animal. Any nucleic acid molecules that contain one or more SNPs of
the present invention can potentially be introduced as a transgene
into the genome of a non-human animal.
Any of the regulatory or other sequences useful in expression
vectors can form part of the transgenic sequence. This includes
intronic sequences and polyadenylation signals, if not already
included. A tissue-specific regulatory sequence(s) can be operably
linked to the transgene to direct expression of the variant protein
in particular cells or tissues.
Methods for generating transgenic animals via embryo manipulation
and microinjection, particularly animals such as mice, have become
conventional in the art and are described in, for example, U.S.
Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat.
No. 4,873,191 by Wagner et al., and in Hogan, B., Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986). Similar methods are used for production of
other transgenic animals. A transgenic founder animal can be
identified based upon the presence of the transgene in its genome
and/or expression of transgenic mRNA in tissues or cells of the
animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene can further be bred to other
transgenic animals carrying other transgenes. A transgenic animal
also includes a non-human animal in which the entire animal or
tissues in the animal have been produced using the homologously
recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced
which contain selected systems that allow for regulated expression
of the transgene. One example of such a system is the cre/loxP
recombinase system of bacteriophage P1 (Lakso et al. PNAS
89:6232-6236 (1992)). Another example of a recombinase system is
the FLP recombinase system of S. cerevisiae (O'Gorman et al.
Science 251:1351-1355 (1991)). If a cre/loxP recombinase system is
used to regulate expression of the transgene, animals containing
transgenes encoding both the Cre recombinase and a selected protein
are generally needed. Such animals can be provided through the
construction of "double" transgenic animals, e.g., by mating two
transgenic animals, one containing a transgene encoding a selected
variant protein and the other containing a transgene encoding a
recombinase.
Clones of the non-human transgenic animals described herein can
also be produced according to the methods described in, for
example, Wilmut, I. et al. Nature 385:810-813 (1997) and PCT
International Publication Nos. WO 97/07668 and WO 97/07669. In
brief, a cell (e.g., a somatic cell) from the transgenic animal can
be isolated and induced to exit the growth cycle and enter G.sub.o
phase. The quiescent cell can then be fused, e.g., through the use
of electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell (e.g., a somatic cell)
is isolated.
Transgenic animals containing recombinant cells that express the
variant proteins described herein are useful for conducting the
assays described herein in an in vivo context. Accordingly, the
various physiological factors that are present in vivo and that
could influence ligand or substrate binding, variant protein
activation, signal transduction, or other processes or
interactions, may not be evident from in vitro cell-free or
cell-based assays. Thus, non-human transgenic animals of the
present invention may be used to assay in vivo variant protein
function as well as the activities of a therapeutic agent or
compound that modulates variant protein function/activity or
expression. Such animals are also suitable for assessing the
effects of null mutations (i.e., mutations that substantially or
completely eliminate one or more variant protein functions).
For further information regarding transgenic animals, see
Houdebine, "Antibody manufacture in transgenic animals and
comparisons with other systems", Curr Opin Biotechnol. 2002
December; 13(6):625-9; Petters et al., "Transgenic animals as
models for human disease", Transgenic Res. 2000; 9(4-5):347-51;
discussion 345-6; Wolf et al., "Use of transgenic animals in
understanding molecular mechanisms of toxicity", J Pharm Pharmacol.
1998 June; 50(6):567-74; Echelard, "Recombinant protein production
in transgenic animals", Curr Opin Biotechnol. 1996 October;
7(5):536-40; Houdebine, "Transgenic animal bioreactors", Transgenic
Res. 2000; 9(4-5):305-20; Pirity et al., "Embryonic stem cells,
creating transgenic animals", Methods Cell Biol. 1998; 57:279-93;
and Robl et al., "Artificial chromosome vectors and expression of
complex proteins in transgenic animals", Theriogenology. 2003 Jan.
1; 59(1):107-13.
Computer-Related Embodiments
The SNPs provided in the present invention may be "provided" in a
variety of mediums to facilitate use thereof. As used in this
section, "provided" refers to a manufacture, other than an isolated
nucleic acid molecule, that contains SNP information of the present
invention. Such a manufacture provides the SNP information in a
form that allows a skilled artisan to examine the manufacture using
means not directly applicable to examining the SNPs or a subset
thereof as they exist in nature or in purified form. The SNP
information that may be provided in such a form includes any of the
SNP information provided by the present invention such as, for
example, polymorphic nucleic acid and/or amino acid sequence
information such as SEQ ID NOS:1-828, SEQ ID NOS:829-1656, SEQ ID
NOS:17,553-18,016, SEQ ID NOS:1657-17,552, and SEQ ID
NOS:18,017-73,085; information about observed SNP alleles,
alternative codons, populations, allele frequencies, SNP types,
and/or affected proteins; or any other information provided by the
present invention in Tables 1-2 and/or the Sequence Listing.
In one application of this embodiment, the SNPs of the present
invention can be recorded on a computer readable medium. As used
herein, "computer readable medium" refers to any medium that can be
read and accessed directly by a computer. Such media include, but
are not limited to: magnetic storage media, such as floppy discs,
hard disc storage medium, and magnetic tape; optical storage media
such as CD-ROM; electrical storage media such as RAM and ROM; and
hybrids of these categories such as magnetic/optical storage media.
A skilled artisan can readily appreciate how any of the presently
known computer readable media can be used to create a manufacture
comprising computer readable medium having recorded thereon a
nucleotide sequence of the present invention. One such medium is
provided with the present application, namely, the present
application contains computer readable medium (CD-R) that has
nucleic acid sequences (and encoded protein sequences) containing
SNPs provided/recorded thereon in ASCII text format in a Sequence
Listing along with accompanying Tables that contain detailed SNP
and sequence information (transcript sequences are provided as SEQ
ID NOS:1-828, protein sequences are provided as SEQ ID
NOS:829-1656, genomic sequences are provided as SEQ ID
NOS:17,553-18,016, transcript-based context sequences are provided
as SEQ ID NOS:1657-17,552, and genomic-based context sequences are
provided as SEQ ID NOS:18,017-73,085).
As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the SNP information of the present invention.
A variety of data storage structures are available to a skilled
artisan for creating a computer readable medium having recorded
thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide/amino acid sequence information of the
present invention on computer readable medium. For example, the
sequence information can be represented in a word processing text
file, formatted in commercially-available software such as
WordPerfect and Microsoft Word, represented in the form of an ASCII
file, or stored in a database application, such as OB2, Sybase,
Oracle, or the like. A skilled artisan can readily adapt any number
of data processor structuring formats (e.g., text file or database)
in order to obtain computer readable medium having recorded thereon
the SNP information of the present invention.
By providing the SNPs of the present invention in computer readable
form, a skilled artisan can routinely access the SNP information
for a variety of purposes. Computer software is publicly available
which allows a skilled artisan to access sequence information
provided in a computer readable medium. Examples of publicly
available computer software include BLAST (Altschul et at, J. Mol.
Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem.
17:203-207 (1993)) search algorithms.
The present invention further provides systems, particularly
computer-based systems, which contain the SNP information described
herein. Such systems may be designed to store and/or analyze
information on, for example, a large number of SNP positions, or
information on SNP genotypes from a large number of individuals.
The SNP information of the present invention represents a valuable
information source. The SNP information of the present invention
stored/analyzed in a computer-based system may be used for such
computer-intensive applications as determining or analyzing SNP
allele frequencies in a population, mapping disease genes,
genotype-phenotype association studies, grouping SNPs into
haplotypes, correlating SNP haplotypes with response to particular
drugs, or for various other bioinformatic, pharmacogenomic, drug
development, or human identification/forensic applications.
As used herein, "a computer-based system" refers to the hardware
means, software means, and data storage means used to analyze the
SNP information of the present invention. The minimum hardware
means of the computer-based systems of the present invention
typically comprises a central processing unit (CPU), input means,
output means, and data storage means. A skilled artisan can readily
appreciate that any one of the currently available computer-based
systems are suitable for use in the present invention. Such a
system can be changed into a system of the present invention by
utilizing the SNP information provided on the CD-R, or a subset
thereof, without any experimentation.
As stated above, the computer-based systems of the present
invention comprise a data storage means having stored therein SNPs
of the present invention and the necessary hardware means and
software means for supporting and implementing a search means. As
used herein, "data storage means" refers to memory which can store
SNP information of the present invention, or a memory access means
which can access manufactures having recorded thereon the SNP
information of the present invention.
As used herein, "search means" refers to one or more programs or
algorithms that are implemented on the computer-based system to
identify or analyze SNPs in a target sequence based on the SNP
information stored within the data storage means. Search means can
be used to determine which nucleotide is present at a particular
SNP position in the target sequence. As used herein, a "target
sequence" can be any DNA sequence containing the SNP position(s) to
be searched or queried.
As used herein, "a target structural motif," or "target motif,"
refers to any rationally selected sequence or combination of
sequences containing a SNP position in which the sequence(s) is
chosen based on a three-dimensional configuration that is formed
upon the folding of the target motif. There are a variety of target
motifs known in the art. Protein target motifs include, but are not
limited to, enzymatic active sites and signal sequences. Nucleic
acid target motifs include, but are not limited to, promoter
sequences, hairpin structures, and inducible expression elements
(protein binding sequences).
A variety of structural formats for the input and output means can
be used to input and output the information in the computer-based
systems of the present invention. An exemplary format for an output
means is a display that depicts the presence or absence of
specified nucleotides (alleles) at particular SNP positions of
interest. Such presentation can provide a rapid, binary scoring
system for many SNPs simultaneously.
One exemplary embodiment of a computer-based system comprising SNP
information of the present invention is provided in FIG. 1. FIG. 1
provides a block diagram of a computer system 102 that can be used
to implement the present invention. The computer system 102
includes a processor 106 connected to a bus 104. Also connected to
the bus 104 are a main memory 108 (preferably implemented as random
access memory, RAM) and a variety of secondary storage devices 110,
such as a hard drive 112 and a removable medium storage device 114.
The removable medium storage device 114 may represent, for example,
a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc. A
removable storage medium 116 (such as a floppy disk, a compact
disk, a magnetic tape, etc.) containing control logic and/or data
recorded therein may be inserted into the removable medium storage
device 114. The computer system 102 includes appropriate software
for reading the control logic and/or the data from the removable
storage medium 116 once inserted in the removable medium storage
device 114.
The SNP information of the present invention may be stored in a
well-known manner in the main memory 108, any of the secondary
storage devices 110, and/or a removable storage medium 116.
Software for accessing and processing the SNP information (such as
SNP scoring tools, search tools, comparing tools, etc.) preferably
resides in main memory 108 during execution.
EXAMPLES
Statistical Analysis of SNP Association with Myocardial Infarction
and Recurrent Myocardial Infarction
Myocardial Infarction Studies (see Table 6)
A case-control genetic study to determine the association of SNPs
in the human genome with MI was carried out using genomic DNA
extracted from 3 independently collected case-control sample
sets.
Study S0012 had 1400 samples, in which patients (cases) had
self-reported history of MI, and controls had no history of MI or
of acute angina lasting more than 1 hour. Study S0028 had 1500
samples, in which patients (cases) had clinical evidence of history
of MI, and controls had no history of MI. Study V0001 had 1288
samples, in which patients (cases) had clinical evidence of history
of MI, and controls had no history of MI. All individuals who were
included in each study had signed a written informed consent form.
The study protocol was IRB approved.
DNA was extracted from blood samples using conventional DNA
extraction methods such as the QIA-amp kit from Qiagen. SNP markers
in the extracted DNA were analyzed by genotyping. While some
samples were individually genotyped, the same samples were also
used for pooling studies, in which DNA from about 50 individuals
was pooled, and allele frequencies were determined in pooled DNA.
For studies S0012(M) and S0012(F), only male or female cases and
controls were used in pooling studies. Genotypes and pool allele
frequencies were obtained using a PRISM 7900HT sequence detection
PCR system (Applied Biosystems, Foster City, Calif.) by
allele-specific PCR, similar to the method described by Germer et
al (Germer S., Holland M. J., Higuchi R. 2000, Genome Res. 10:
258-266). Primers for the allele-specific PCR reactions are
provided in Table 5.
Summary statistics for demographic and environmental traits,
history of vascular disease, and allele frequencies for the tested
SNPs were obtained and compared between cases and controls. No
multiple testing corrections were made.
Tests of association were calculated for both non-stratified and
stratified settings: 1) Fisher's exact test of allelic association,
and 2) asymptotic chi-square test of genotypic association, taking
two different modes of inheritance into account (dominant, and
recessive).
Effect sizes were estimated through allelic odds ratios, including
95% confidence intervals. The reported Allele1 may be
under-represented in cases (with a lower allele frequency in cases
than in controls, indicating that the reported Allele1 is
associated with decreased risk and the other allele is a risk
factor for disease) or over-represented in cases (indicating that
the reported Allele1 is a risk factor in the development of
disease).
A SNP was considered to be a significant genetic marker if it
exhibited a p-value <0.05 in the allelic association test or in
any of the 2 genotypic tests (dominant, recessive). The association
of a marker with MI was considered replicated if the marker
exhibited an allelic or genotypic association test p-value <0.05
in one of the sample sets and the same test and strata (or
substrata) were significant (p<0.05) in another independent
sample set.
An example of a replicated marker, where the reported Allele1 is
associated with decreased risk for MI, is hCV8851074 (Table 6).
hCV8851074 shows significant association with all individuals
(strata="ALL") of study S0028 and the non-smoking strata
(Stratification="SMOKE", Strata="N") of study S0012. The odds ratio
in both studies is less than 1 (0.84 and 0.78 respectively), using
the same allele (Allele1="A") for analysis.
An example of a replicated marker, where the reported Allele1 is
associated with increased risk for MI, is hCV2716008 (Table 6).
hCV2716008 shows significant association with all individuals
(strata="ALL") of study S0028 and all males of study S0012
(strata="ALL", study="S0012(M)"). The odds ratio in both studies is
greater than 1 (1.42 and 1.32 respectively), using the same allele
(Allele1="C") for analysis.
Recurrent Myocardial Infarction (RMI) Studies (See Table 7)
In order to identify genetic markers associated with recurrent
myocardial infarction (RMI), samples from the Cholesterol and
Recurrent Events (CARE) study were genotyped utilizing 864 assays
for functional SNPs in 500 candidate genes. A well-documented MI
was one of the enrollment criteria for the CARE study. Patients
were followed up for 5 years and rates of recurrent MI were
recorded in Pravastatin treated and placebo groups.
In the initial analysis (CARE), SNP genotype frequencies were
compared in a group of 264 patients who had another MI (second,
third, or fourth) during the 5 years of CARE follow-up (cases)
versus the frequencies in the group of 1255 CARE patients who had
not experienced a second MI (controls).
To replicate the initial findings, a second group of 394 CARE
patients were analyzed who had a history of an MI prior to the MI
at CARE enrollment but who had not experienced an MI during trial
follow-up (cases), and 1221 CARE MI patients without a second MI
were used as controls (Pre-CARE Study). No patients from the CARE
Study were used in the Pre-CARE Study.
The SNPs replicated between CARE and Pre-CARE Studies were also
tested for primary MI in study S0012 (UCSF). Study S0012 had 1400
samples. MI patients (cases) in this study had a self-reported
history of MI, and controls had no history of MI or of acute angina
lasting more than 1 hour. Allele frequencies in this study were
detected in pooling experiments, in which DNA from about 50
individuals was pooled, and allele frequencies were determined in
pooled DNA. For study S0012, only male cases and controls were used
in pooling studies.
DNA was extracted from blood samples using conventional DNA
extraction methods like the QIA-amp kit from Qiagen. Genotypes were
obtained on a PRISM 7900HT sequence detection PCR system (Applied
Biosystems) by allele-specific PCR, similar to the method described
by Germer et al (Germer S., Holland M. J., Higuchi R. 2000, Genome
Res. 10: 258-266). Primers for the allele-specific PCR reactions
are provided in Table 5.
Statistical analysis was done using asymptotic chi square test for
allelic, dominant, or recessive association, or Armitage trend test
for additive genotypic association in the non-stratified as well as
in the strata. Replicated SNPs are provided in Table 7. A SNP is
considered replicated if the at-risk alleles in both sample sets
are identical, the p-values are less than 0.05 in both sample sets,
and the significant association is seen in the same stratum in both
sets or one stratum is inclusive of the other.
Effect sizes were estimated through allelic odds ratios and odds
ratios for dominant and recessive models, including 95% confidence
intervals. Homogeneity of Cochran-Mantel-Haenszel odds ratios was
tested across different strata using the Breslow-Day test. A SNP
was considered to be a significant genetic marker if it exhibited a
p-value <0.05 in the allelic association test or in any of the 3
genotypic tests (dominant, recessive, additive). Haldane Odds
Ratios were used if either case or control count was zero. SNPs
with significant HWE violations in both cases and controls
(p<1.times.10.sup.-4 in both tests) were not considered for
further analysis, since significant deviation from HWE in both
cases and controls for individual markers can be indicative of
genotyping errors. The association of a marker with RMI was
considered replicated if the marker exhibits an allelic or
genotypic association test p-value <0.05 in one of the sample
sets and the same test and strata are significant (p<0.05) in
either one or two other independent sample sets.
All publications and patents cited in this specification are herein
incorporated by reference in their entirety. Various modifications
and variations of the described compositions, methods and systems
of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments and certain working examples, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the above-described modes for carrying out the
invention that are obvious to those skilled in the field of
molecular biology, genetics and related fields are intended to be
within the scope of the following claims.
TABLE-US-00006 TABLE 5 Sequence A Sequence B Sequence C hCV Alleles
(allele-specific primer) (allele-specific primer) (common primer)
hCV1022614 G/A CTGCAGCCTCTCCTACG CCTGCAGCCTCTCCTACA
GATTCCCCATCGGTCATAA (SEQ ID NO: 73086) (SEQ ID NO: 73087) (SEQ ID
NO: 73088) hCV1026583 A/G GGAAGTTGAGATTCTTTCAGAA
GGAAGTTGAGATTCTTTCAGAG AGTGATTGGAAAT- CCATATTTACTT (SEQ ID NO:
73089) (SEQ ID NO: 73090) (SEQ ID NO: 73091) hCV1065191 G/T
GCCCCACTTTTGCATG AGCCCCACTTTTGCATT ACCCCTGCACAGTTTAGAAC (SEQ ID NO:
73092) (SEQ ID NO: 73093) (SEQ ID NO: 73094) hCV1085595 T/C
GGTGCTCCACCTGGT GTGCTCCACCTGGC GGAGTTCGAACCTAAAGACGTAT (SEQ ID NO:
73095) (SEQ ID NO: 73096) (SEQ ID NO: 73097) hCV1085600 C/G
AGCTGTTCGTGTTCTATGATC AGCTGTTCGTGTTCTATGATG GAAGTCAACAGTGAA-
CATGTGA (SEQ ID NO: 73098) (SEQ ID NO: 73099) (SEQ ID NO: 73100)
hCV11159941 G/A CCCGTTGGTTCGAAG CCCGTTGGTTCCGAAAA
TGAATAGCCATTAGAAAAAACTGT- (SEQ ID NO: 73101) (SEQ ID NO: 73102)
(SEQ ID NO: 73103) hCV1129436 C/A TTCCAGGGTATATCTCAGAGC
GTTCCAGGGTATATCTCAGAGA TTGAAAGAGTGTGA- GCAAGATC (SEQ ID NO: 73104)
(SEQ ID NO: 73105) (SEQ ID NO: 73106) hCV11359098 G/C
CAAAATGTAGAAGGTTCATATGAG CAAAATGTAGAAGGTTCATATGAC GAGCTGTG-
TGTTTCTTTGTTCTA (SEQ ID NO: 73107) (SEQ ID NO: 73108) (SEQ ID NO:
73109) hCV11442703 C/T CCCAGGGCTCCTGAC CCCAGGGCTCCTGAT
AAAAAGCCCTTTGGTATTGTATA (SEQ ID NO: 73110) (SEQ ID NO: 73111) (SEQ
ID NO: 73112) hCV11482579 T/A GTTGAAGGGAAGTTCAGCAT
TTGAAGGGAAGTTCAGCAA TCACGGAGGACAGGTAG- AAT (SEQ ID NO: 73113) (SEQ
ID NO: 73114) (SEQ ID NO: 73115) hCV11482766 G/A GCGCACCCAGGTCAG
GCGCACCCAGGTCAA CCACGTTCTGGTCGATCTT (SEQ ID NO: 73116) (SEQ ID NO:
73117) (SEQ ID NO: 73118) hCV11482773 A/C CTGCTGCTGCTCCTGA
TGCTGCTGCTCCTGC ACTTGAGCTTCCTGGAGAAG (SEQ ID NO: 73119) (SEQ ID NO:
73120) (SEQ ID NO: 73121) hCV11484594 C/T CCACAGCGAGGCTTTTC
CCACAGCGAGGCTTTTT GCACTAATGTGATCGTTGAAAA- (SEQ ID NO: 73122) (SEQ
ID NO: 73123) (SEQ ID NO: 73124) hCV11486078 C/G AGCCCCAGAACCTGC
AGCCCCAGAACCTGG GGGCTGGGCTTGTAGAATA (SEQ ID NO: 73125) (SEQ ID NO:
73126) (SEQ ID NO: 73127) hCV11506744 T/G GAAAAGGAGGATGAAGATGTCT
AAAGGAGGATGAAGATGTCG CACCATGCTCTGCA- AAGAC (SEQ ID NO: 73128) (SEQ
ID NO: 73129) (SEQ ID NO: 73130) hCV11592758 T/C
CATCCAACAGCTCTTCTATCAT CATCCAACAGCTCTTCTATCAC CAAACATCCGAG- GACAAG
(SEQ ID NO: 73131) (SEQ ID NO: 73132) (SEQ ID NO: 73133)
hCV11628130 A/T CTGCCCTCTTTTTAGCAGA CTGCCCTCTTTTTAGCAGT
CCCTTTCTCATTCATTCA- TTTT (SEQ ID NO: 73134) (SEQ ID NO: 73135) (SEQ
ID NO: 73136) hCV11655948 A/G GACGTCTTCCAGTACCA ACGTCTTCCAGTACCG
TCTTCCTCGCTCAGAAT (SEQ ID NO: 73137) (SEQ ID NO: 73138) (SEQ ID NO:
73139) hCV11655953 T/C AGCATTGCCGTCCT GCATTGCCGTCCC
CCATGGGTCAAAGAACA (SEQ ID NO: 73140) (SEQ ID NO: 73141) (SEQ ID NO:
73142) hCV11668930 T/C CCAGATCCAGTTTTCTAGCTAGCAT
CCAGATCCAGTTTTCTAGCAC CCCTTCCTCC- AGCATTATC (SEQ ID NO: 73143) (SEQ
ID NO: 73144) (SEQ ID NO: 73145) hCV11689917 T/C CTGTGAGTGGGCCTTCAT
CTGTGAGTGGGCCTTCAC GGAGCCCCGCTTCAT (SEQ ID NO: 73146) (SEQ ID NO:
73147) (SEQ ID NO: 73148) hCV11689926 C/T GAAGGCCCACTCACAGAC
GAAGGCCCACTCACAGAT CGGACCCGGAGACTG (SEQ ID NO: 73149) (SEQ ID NO:
73150) (SEQ ID NO: 73151) hCV11689930 A/G TCACAGACTGACCGAGTGA
CACAGACTGACCGAGTGG CGGACCCGGAGACTGT (SEQ ID NO: 73152) (SEQ ID NO:
73153) (SEQ ID NO: 73154) hCV11696920 A/G GTCTTTAGAAGCCTCTTCAGAATA
CTTTAGAAGCCTCTTCAGAATG CGGCTTTGGC- CTACAAG (SEQ ID NO: 73155) (SEQ
ID NO: 73156) (SEQ ID NO: 73157) hCV11758801 C/G
AGTACCTCTTGGTCTCTCTCC AGTACCTCTTGGTCTCTCTCG GCATGTTGTGTTTC-
TGATTGTAC (SEQ ID NO: 73158) (SEQ ID NO: 73159) (SEQ ID NO: 73160)
hCV11764545 G/T ACGAAGCTTCCGAGGAAG CGAAGCTTCCGAGGAAT
GACACCGGACGAGAGAGAC (SEQ ID NO: 73161) (SEQ ID NO: 73162) (SEQ ID
NO: 73163) hCV11789692 G/C ATCCCCCACAGATCCAG ATCCCCCACAGATCCAC
CCAGCTGGACCCAGTAAG (SEQ ID NO: 73164) (SEQ ID NO: 73165) (SEQ ID
NO: 73166) hCV11889257 T/C CTCTCTTTCTAGAAGAAATT
TCTCTTTCTAGAAACTGAAGAAATC GGGCAGGGCTA- GGAGTAG (SEQ ID NO: 73167)
(SEQ ID NO: 73168) (SEQ ID NO: 73169) hCV11937023 C/T
GGATATGAGTTGGACATGAAGAC GGATATGAGTTGGACATGAAGAT CAGGTTTTGG-
TGGGAGAAC (SEQ ID NO: 73170) (SEQ ID NO: 73171) (SEQ ID NO: 73172)
hCV11951095 T/C CGTGACCCTGCCGT CGTGACCCTGCCGC GGGCCAGCATGTGGAC (SEQ
ID NO: 73173) (SEQ ID NO: 73174) (SEQ ID NO: 73175) hCV11955747 T/G
AAAGGGAAGGAGGTTACTTACT AGGGAAGGAGGTTACTTACG TCCTCTGTGGAGAG- GGATAC
(SEQ ID NO: 73176) (SEQ ID NO: 73177) (SEQ ID NO: 73178)
hCV11972321 G/A GGTTCTGACATGACTGTGACAG GTTCTGACATGACTGTGACAA
CCCCAACCCAAGG- TTTAC (SEQ ID NO: 73179) (SEQ ID NO: 73180) (SEQ ID
NO: 73181) hCV11975283 A/C GGGCTGGACATTGCAA GGGCTGGACATTGCAC
CCAGATTAGCCTTAACTCTGTTAA- C (SEQ ID NO: 73182) (SEQ ID NO: 73183)
(SEQ ID NO: 73184) hCV1202883 G/A GCGTGATGATGAAATCGG
GCGTGATGATGAAATCGA AGCCTCTCCTGACTGTCATC (SEQ ID NO: 73185) (SEQ ID
NO: 73186) (SEQ ID NO: 73187) hCV12029981 A/G ACGAGAGCATCATCTGCA
CGAGAGCATCATCTGCG CCAGACATTGCAGTTGAAGTC- (SEQ ID NO: 73188) (SEQ ID
NO: 73189) (SEQ ID NO: 73190) hCV1207994 T/G GCAGCAGTCGCCCTT
GCAGCAGTCGCCCTG CATTTTGCTGATGTTTGTTTCTAG (SEQ ID NO: 73191) (SEQ ID
NO: 73192) (SEQ ID NO: 73193) hCV12083298 A/G CAGAACGCTGGGAAACA
AGAACGCTGGGAAACG CTCCCCACCTGTTGCTC (SEQ ID NO: 73194) (SEQ ID NO:
73195) (SEQ ID NO: 73196) hCV12114319 T/C GACACTGCCCTCATCGT
CACTGCCCTCATCGC CCTGTCCTTGAGGTCTGATC (SEQ ID NO: 73197) (SEQ ID NO:
73198) (SEQ ID NO: 73199) hCV1212713 T/C CCCAGTGGGTCCT
CCCAGTGGGTCCC TGCCATCGTTGTTTTG (SEQ ID NO: 73200) (SEQ ID NO:
73201) (SEQ ID NO: 73202) hCV1260328 T/C TCCACGTGGACCAGGT
CCACGTGGACCAGGC GCCCAGGTATTTCATCAGC (SEQ ID NO: 73203) (SEQ ID NO:
73204) (SEQ ID NO: 73205) hCV1276216 T/C TCTTATCAGTCTTGGACAAGAACT
TTATCAGTCTTGGACAAGAACC CTCTTGCTTTC- TGTCTTCATAGAC (SEQ ID NO:
73206) (SEQ ID NO: 73207) (SEQ ID NO: 73208) hCV1345898 C/T
CAGTTTTCCATGGGTTCTACTAC CAGTTTTCCATGGGTTCTACTAT TTATGAAATGG-
TACAGACAAGTGAT (SEQ ID NO: 73209) (SEQ ID NO: 73210) (SEQ ID NO:
73211) hCV1376137 A/G CTCCATCATTGCAGACCA TCCATCATTGCAGACCG
CCAATTCCCCTGATGTTAAA (SEQ ID NO: 73212) (SEQ ID NO: 73213) (SEQ ID
NO: 73214) hCV1376266 T/A CGCCCCTCCCGCT CGCCCCTCCCGCA
TGATCCCTCCCTTGGATA (SEQ ID NO: 73215) (SEQ ID NO: 73216) (SEQ ID
NO: 73217) hCV1376342 C/T ACTCACCAGTTGTGAAGACTTC
TACTCACCAGTTGTGAAGACTTT TCAGGGAGCCTA- GATATCTCA (SEQ ID NO: 73218)
(SEQ ID NO: 73219) (SEQ ID NO: 73220) hCV1385736 T/C
TGGTGTCAGGTCCCCTT TGGTGTCAGGTCCCCTC GCAGCCACCTTGACACTC (SEQ ID NO:
73221) (SEQ ID NO: 73222) (SEQ ID NO: 73223) hCV1387523 T/C
TGGGGTTGGGGTTCT GGGGTTGGGGTTCC TGTCCCTGTCCTCCTTCAG (SEQ ID NO:
73224) (SEQ ID NO: 73225) (SEQ ID NO: 73226) hCV1403468 T/G
ACTGGCCCCTTGCAT ACTGGCCCCTTGCAG AGGAGGGAACCAAACCTTA (SEQ ID NO:
73227) (SEQ ID NO: 73228) (SEQ ID NO: 73229) hCV1419869 G/C
AGTTGTCAGTGCCTCTGTTG AGTTGTCAGTGCCTCTGTTC ACAACCCTTGAATGAGA- GAATAC
(SEQ ID NO: 73230) (SEQ ID NO: 73231) (SEQ ID NO: 73232) hCV1466546
A/G TCTGGCTTCCGGGAA TCTGGCTTCCGGGAG CGTAGCTGTTGACCATCATTA (SEQ ID
NO: 73233) (SEQ ID NO: 73234) (SEQ ID NO: 73235) hCV1186426 A/G
CACAGGCTATGACCTCAACA ACAGGCTATGACCTCAACG AACCATGGAGCTACTCTT- CTGTA
(SEQ ID NO: 73236) (SEQ ID NO: 73237) (SEQ ID NO: 73238) hCV14938
T/C AGGTGGAGATTCTCAACAGAT AGGTGGAGATTCTCAACAGAC AGGCTGTTCTCATGACA-
TACAT (SEQ ID NO: 73239) (SEQ ID NO: 73240) (SEQ ID NO: 73241)
hCV1552900 A/G GGGATGGAAGAGCTTCA GGGATGGAAGAGCTTCG
CTGCAGCCTTCCTCTGAC (SEQ ID NO: 73242) (SEQ ID NO: 73243) (SEQ ID
NO: 73244) hCV15751934 T/C ACTATGAGAGCATCATGTGT CTATGAGAGCATCATGTGC
ACTGAGTCCTGAAGAAA- AATCAG (SEQ ID NO: 73245) (SEQ ID NO: 73246)
(SEQ ID NO: 73247) hCV15760070 A/T TGTCCAGATCCACATAGAACA
TTGTCCAGATCCACATAGAACT CTTTATGCAGCGG- ACCAT (SEQ ID NO: 73248) (SEQ
ID NO: 73249) (SEQ ID NO: 73250) hCV15851292 C/T TCCCCCGAGAAGAAGAC
TCCCCCGAGAAGAAGAT TGCTGTCAACCCTCTCTCTT (SEQ ID NO: 73251) (SEQ ID
NO: 73252) (SEQ ID NO: 73253) hCV15853800 T/G ACCTGGTGGCACATCTAATAT
CCTGGTGGCACATCTAATAG TGTGGAATGTGGAAA- CAATACT (SEQ ID NO: 73254)
(SEQ ID NO: 73255) (SEQ ID NO: 73256) hCV15859395 G/T
GCCTGCATTACTCAAGGG TGCCTGCATTACTCAAGGT CAGAGAGGGTCAGGAGTTG- A (SEQ
ID NO: 73257) (SEQ ID NO: 73258) (SEQ ID NO: 73259) hCV15859649 G/A
CTGCCATGATGTTCACG CCTGCCATGATGTTCACA GATGGCTGCATAGCAGTAGAT- (SEQ ID
NO: 73260) (SEQ ID NO: 73261) (SEQ ID NO: 73262) hCV15869253 G/T
CTACTGCCCCGACCTG CTACTGCCCCGACCTT GTCATGGCGCTACTAGATGTAT (SEQ ID
NO: 73263) (SEQ ID NO: 73264) (SEQ ID NO: 73265) hCV15878378 T/C
TGCCTGGTGAGGGAGAT TGCCTGGTGAGGGAGAC CACACACAAGAAAGTAAAACAT- GAA
(SEQ ID NO: 73266) (SEQ ID NO: 73267) (SEQ ID NO: 73268)
hCV15966517 A/G CACCTTCCAGCCGGTA ACCTTCCAGCCGGTG
TGAGGTAGCCCAGTGACTTC (SEQ ID NO: 73269) (SEQ ID NO: 73270) (SEQ ID
NO: 73271) hCV15968121 T/C TCAAAATTCATGGTCACTTTAAT
TCAAAATTCATGGTCACTTTAAC CCTCTTCCAT- GCACTCACTT (SEQ ID NO: 73272)
(SEQ ID NO: 73273) (SEQ ID NO: 73274) hCV15976768 C/G
TGGATGTTGGAACAATTGAC TGGATGTTGGAACAATTGAG CCAGCCAGCAAAGATA- CATAC
(SEQ ID NO: 73275) (SEQ ID NO: 73276) (SEQ ID NO: 73277)
hCV15976971 T/C GAGCCCTGCGCACT AGCCCTGCGCACC GGCACGATTGCTCTTAGTGT
(SEQ ID NO: 73278) (SEQ ID NO: 73279) (SEQ ID NO: 73280)
hCV16033535 G/T GGTCGTCCTGCACTTCG TGGTCGTCCTGCACTTCT
CCAGCTCCCCTCTCTTTTC (SEQ ID NO: 73281) (SEQ ID NO: 73282) (SEQ ID
NO: 73283) hCV16072719 A/G TCACCTGCTTTTGCCA CACCTGCTTTTGCCG
GGCCCAACCCAGTGAC (SEQ ID NO: 73284) (SEQ ID NO: 73285) (SEQ ID NO:
73286) hCV1608777 G/A CAAGAGGATTTTTATGGAATGG
ACAAGAGGATTTTTATGGAATGA GGTGGTGAGGCT- TTGAGTATA (SEQ ID NO: 73287)
(SEQ ID NO: 73288) (SEQ ID NO: 73289) hCV16089120 T/C
CCACATTCTCTCTCCTT CCCACATTCTCTCTCCTC TGAGGAGCGTGGGTGAC (SEQ ID NO:
73290) (SEQ ID NO: 73291) (SEQ ID NO: 73292) hCV16165996 C/T
CTGAGGCCTATGTCCTC CTGAGGCCTATGTCCTT AGCTCTCCTTTGTTGCTACTG (SEQ ID
NO: 73293) (SEQ ID NO: 73294) (SEQ ID NO: 73295)
hCV16166043 T/C CGGTTGAAGTCCTTGAT CGGTTGAAGTCCTTGAC
GGTTGTGCAGAGAGACATGTGA- (SEQ ID NO: 73296) (SEQ ID NO: 73297) (SEQ
ID NO: 73298) hCV16170641 G/C TCTGCTTAAATATGGCTTGTG
TTCTGCTTAAATATGGCTTGTC GCAGTTGTTAAGG- ACAGAAATACTT (SEQ ID NO:
73299) (SEQ ID NO: 73300) (SEQ ID NO: 73301) hCV16170651 C/T
AACAAACGTACCATTGAGGC GAAACAAACGTACCATTGAGGT TCCACGCTGATCTG- AAGATAC
(SEQ ID NO: 73302) (SEQ ID NO: 73303) (SEQ ID NO: 73304)
hCV16170900 T/C CGCACACCAGGTTCTCAT CGCACACCAGGTTCTCAC
GCAACTACCTGGGCCACTAT- A (SEQ ID NO: 73305) (SEQ ID NO: 73306) (SEQ
ID NO: 73307) hCV16170982 C/G CCCCCACTCTCCAGC CCCCCACTCTCCAGG
GGCAAAAGCACTGTGAAGA (SEQ ID NO: 73308) (SEQ ID NO: 73309) (SEQ ID
NO: 73310) hCV16171764 G/A CCCCAACCACCACG GCCCCAACCACCACA
GCGTTCCGGGAAGACTT (SEQ ID NO: 73311) (SEQ ID NO: 73312) (SEQ ID NO:
73313) hCV16172098 C/T GACACACAGGGTGGCTC GACACACAGGGTGGCTT
TCTGGTCTGCCTCAGGTAAC (SEQ ID NO: 73314) (SEQ ID NO: 73315) (SEQ ID
NO: 73316) hCV16172249 G/C ACTGTAATTTTTTTAAGGTCCTG
ACTGTAATTTTTTTAAAGGTCCTC GGATGTATA- TCATCTATCTTCACAGTA (SEQ ID NO:
73317) (SEQ ID NO: 73318) TAT (SEQ ID NO: 73319) hCV16172571 T/C
GGTACCATGGACTGTACTCACT GTACCATGGACTGTACTCACC AGGTTGGTTCTGG- AGATGAC
(SEQ ID NO: 73320) (SEQ ID NO: 73321) (SEQ ID NO: 73322)
hCV16181123 C/A AATTCAAGACAGTGCAACATC TAAATTCAAGACAGTGCAACATA
CACACACCGCAC- TCTAATTACT (SEQ ID NO: 73323) (SEQ ID NO: 73324) (SEQ
ID NO: 73325) hCV16191372 C/T AGGTCGTGTTGCCGC GAGGTCGTGTTGCCGT
GCCTCTCACCACTTTCTGTAAG (SEQ ID NO: 73326) (SEQ ID NO: 73327) (SEQ
ID NO: 73328) hCV16192174 G/A GAGCACCTTAACTATAGATGGTG
TGAGCACCTTAACTATAGATGGTA CTTGTCAAG- GCACAGAATAATT (SEQ ID NO:
73329) (SEQ ID NO: 73330) (SEQ ID NO: 73331) hCV16196014 G/C
TGAAGAAGCTAAGGATTGAGG TGAAGAAGCTAAGGATTGAGC CTCTCCCTGGCTGA- GTTG
(SEQ ID NO: 73332) (SEQ ID NO: 73333) (SEQ ID NO: 73334)
hCV16266313 T/C CACGGCGCACTTTCTT CACGGCGCACTTTCTC
TGTTTTTTCCTTTGTCATCTTATC- TA (SEQ ID NO: 73335) (SEQ ID NO: 73336)
(SEQ ID NO: 73337) hCV16273460 G/A GCACTCTTGGACAAGCG
TGCACTCTTGGACAAGCA AATGACATCCCCTATCTTCTG- (SEQ ID NO: 73338) (SEQ
ID NO: 73339) (SEQ ID NO: 73340) hCV16276495 C/T
GTACCTTCACCCATGGAAC GTACCTTCACCCATGGAAT TCACTTTCTGTTGATTAC- ATGAGA
(SEQ ID NO: 73341) (SEQ ID NO: 73342) (SEQ ID NO: 73343) hCV1639938
T/G AGTGGAGCTTCAGGGCT TGGAGCTTCAGGGCG CAGTGGAGACAGAGGATGTTTAC (SEQ
ID NO: 73344) (SEQ ID NO: 73345) (SEQ ID NO: 73346) hCV1662671 A/G
CAGCCAAGAGCAGGACA AGCCAAGAGCAGGACG CCCAAGACACGTTCAGAAAT (SEQ ID NO:
73347) (SEQ ID NO: 73348) (SEQ ID NO: 73349) hCV1841898 T/C
TCTCCCTCCTGTTCCTTGT TCCCTCCTGTTCCTTGC GCAATGTGCTGTGAATGAAG (SEQ ID
NO: 73350) (SEQ ID NO: 73351) (SEQ ID NO: 73352) hCV1842400 T/C
TTGGTACCTGGCTCTCT TGGTACCTGGCTGTCC AAACTTCTTAGGACAGAGTGATTA- GA
(SEQ ID NO: 73353) (SEQ ID NO: 73354) (SEQ ID NO: 73355) hCV1923359
G/C CCTCAGGCACCGAGAG CCTCAGGCACCGAGAC CTGGACCTCCTGATGATCTC (SEQ ID
NO: 73356) (SEQ ID NO: 73357) (SEQ ID NO: 73358) hCV1985481 T/C
CTGGCTGCTCCCTGAT CTGGCTGCTCCCTGAC GCCTGACTGGCTGATCTC (SEQ ID NO:
73359) (SEQ ID NO: 73360) (SEQ ID NO: 73361) hCV1998030 T/C
GGTGCAGAACCT GGGTGCAGAACCC CAGACCGGCCCACTTG (SEQ ID NO: 73362) (SEQ
ID NO: 73363) (SEQ ID NO: 73364) hCV2033404 T/C CCATCTTCTACGCACTGT
ATCTTCTACGCACTGC CAGCATAGCCAGGAAGAAGA (SEQ ID NO: 73365) (SEQ ID
NO: 73366) (SEQ ID NO: 73367) hCV2033405 G/A GAGCGGGAGCAACG
TGAGCGGGAGCAACA TGGAGCTGCAGGTGATC (SEQ ID NO: 73368) (SEQ ID NO:
73369) (SEQ ID NO: 73370) hCV2038 G/A CACGGCGGTCATGTG
CCACGGCGGTCATGTA GTTTTGTGGGAGGAAAGAG (SEQ ID NO: 73371) (SEQ ID NO:
73372) (SEQ ID NO: 73373) hCV2045908 C/A CAGTTGTACCCCTACAATAATGTAC
CAGTTGTACCCCTACAATAATGTAA TCGGTTT- CTCTTCATTCATTC (SEQ ID NO:
73374) (SEQ ID NO: 73375) (SEQ ID NO: 73376) hCV210485 C/G
GGCGTTCAGTTTTTAGCC GGCGTTCAGTTTTTAGCG CAATACGCCAGCAAAATACC (SEQ ID
NO: 73377) (SEQ ID NO: 73378) (SEQ ID NO: 73379) hCV2143205 T/C
TGTCGAATGGGAGTCTTCTT TGTCGAATGGGAGTCTTCTC GGAAGAAACAGCTACCC- AGA
(SEQ ID NO: 73380) (SEQ ID NO: 73381) (SEQ ID NO: 73382) hCV2146578
C/T CCTCCTAGAGAAGATCTGACAC CCTCCTAGAGAAGATCTGCAT AAGGCCCCTCTTCT-
GTCT (SEQ ID NO: 73383) (SEQ ID NO: 73384) (SEQ ID NO: 73385)
hCV2188895 A/G AGAGAATGTTACCTCTCCTGA GAGAATGTTACCTCTCCTGG
TTCTCCTGGGTCAGAT- TCTC (SEQ ID NO: 73386) (SEQ ID NO: 73387) (SEQ
ID NO: 73388) hCV2200985 G/C GCGCACCAGCTTCAG GCGCACCAGCTTCAC
TGTAATACATGATTTTCAGACACAC (SEQ ID NO: 73389) (SEQ ID NO: 73390)
(SEQ ID NO: 73391) hCV22271841 C/T CATCACGGAGATCCACC
ATCATCACGGAGATCCACT TCAGCTCCAAGGAGATTCTT- AG (SEQ ID NO: 73392)
(SEQ ID NO: 73393) (SEQ ID NO: 73394) hCV22271999 G/A
GAGCACCTTAACTATAGATGGTG TGAGCACCTTAACTATAGATGGTA CTTGTCAAG-
GCACAGAATAATT (SEQ ID NO: 73395) (SEQ ID NO: 73396) (SEQ ID NO:
73397) hCV22272267 T/C CTGGCAGCGAATGTTAT CTGGCAGCGAATGTTAC
CCTCTAGAAAGAAAATGGACTG- TAT (SEQ ID NO: 73398) (SEQ ID NO: 73399)
(SEQ ID NO: 73400) hCV22273419 C/T ACTCCTTTGGACTGGC
TGACTCCTTTGGACTGGT TCTTAAATGCTGTGGAATTGTG- (SEQ ID NO: 73401) (SEQ
ID NO: 73402) (SEQ ID NO: 73403) hCV22274307 C/T
GACCTACGCTCCTTCATCTAAC GACCTACGCTCCTTCATCTAAT CAATGTGATTAC-
CTCAAAATAATATCT (SEQ ID NO: 73404) (SEQ ID NO: 73405) AC (SEQ ID
NO: 73406) hCV22274425 C/T AGAGAGGGTGAAGGTGC GAGAGAGGGTGAAGGTGT
CCCTTTAAGGCTGTTATCTGA- C (SEQ ID NO: 73407) (SEQ ID NO: 73408) (SEQ
ID NO: 73409) hCV22274540 C/T ACCACTTCTATCCGAGTAGC
GACCACTTCTATCCGAGTAGT GGGAAAAATGTGTGT- CTTTTAATTA (SEQ ID NO:
73410) (SEQ ID NO: 73411) (SEQ ID NO: 73412) hCV22274624 G/A
CCCTACAGAGGATGTCAG CCCTACAGAGGATGTCAA CAGAGCCTCCCTTGTCAC (SEQ ID
NO: 73413) (SEQ ID NO: 73414) (SEQ ID NO: 73415) hCV2253681 T/C
CCTGTCGGGGGAGAT CCTGTCGGGGGAGAC GAAGCGGAGCCTGAGAA (SEQ ID NO:
73416) (SEQ ID NO: 73417) (SEQ ID NO: 73418) hCV2259750 A/T
AGAAACTGGCTCTGAAGACA CAGAAACTGGCTCTGAAGACT GAAAGTGGGCATGGGT- ATAC
(SEQ ID NO: 73419) (SEQ ID NO: 73420) (SEQ ID NO: 73421) hCV2259921
T/C CTTTCGAAGTTTCAGTTGAACT TTCGAAGTTTCAGTTGAACC GTTTTCCAGCTTGCA-
TGTC (SEQ ID NO: 73422) (SEQ ID NO: 73423) (SEQ ID NO: 73424)
hCV2303890 T/G CACTATCGAAGTCCCCAAAT CACTATCGAAGTCCCCAAAG
CATCTGGTTTTTGACAA- TCATTATA (SEQ ID NO: 73425) (SEQ ID NO: 73426)
(SEQ ID NO: 73427) hCV2310409 A/T CTCAGGGAGGGAGAGAGA
CTCAGGAGGGAGAGAGT ACAGCTCAGGCAGAAACTG (SEQ ID NO: 73428) (SEQ ID
NO: 73429) (SEQ ID NO: 73430) hCV2415786 A/G CTCAGCTGAACCTGGCTA
CAGCTGAACCTGGCTG GAAACACCTCCTCCATCTTC (SEQ ID NO: 73431) (SEQ ID
NO: 73432) (SEQ ID NO: 73433) hCV2443247 G/T CCAGAATGCCTTCATCG
GCCAGAATGCCTTCATCT GCATCACAGCTTGCTTCTATAA- (SEQ ID NO: 73434) (SEQ
ID NO: 73435) (SEQ ID NO: 73436) hCV2462424 C/T
CTTATGAGATTTTCTGTCCAGTC CTTATGAGATTTTCTGTCCAGTT TGGAATTGCAG-
ATGAACAAG (SEQ ID NO: 73437) (SEQ ID NO: 73438) (SEQ ID NO: 73439)
hCV2531086 A/G GCCCCCCTCTCTGAAGA CCCCCCTCTCTGAAGG
CCAGTTCGTGGTATGTTCATCT (SEQ ID NO: 73440) (SEQ ID NO: 73441) (SEQ
ID NO: 73442) hCV2531732 T/C CCACTGTTGCCATGAT CCACTGTTGCCATGAC
CCTTCCGGCTCCATAAG (SEQ ID NO: 73443) (SEQ ID NO: 73444) (SEQ ID NO:
73445) hCV2531795 C/G CTCTCAGCTCAAGCCTCC TCTCAGCTCAAGCCTCG
CCTGCCCACCTGTCTCT (SEQ ID NO: 73446) (SEQ ID NO: 73447) (SEQ ID NO:
73448) hCV2532034 C/T CTGAAGCGCAACCATAAC CTGAAGCGCAACCATAAT
TCCCATAGAAAAATGCACTAA- G (SEQ ID NO: 73449) (SEQ ID NO: 73450) (SEQ
ID NO: 73451) hCV25472003 A/C GGTAAAGCTCCAGCACTGTA
GGTAAAGCTCCAGCACTGTC TCAGCAGCAGGTTCAC- ATAA (SEQ ID NO: 73452) (SEQ
ID NO: 73453) (SEQ ID NO: 73454) hCV25472673 C/T TGGGCTCCATCCCAC
TGGGCTCCATCCCAT CCAATTCTTTTTCTTCTTTCAGTT (SEQ ID NO: 73455) (SEQ ID
NO: 73456) (SEQ ID NO: 73457) hCV25473150 T/C CCCACATTCTCTCTCCTT
CCCACATTCTCTCTCCTC TGAGGAGCGTGGGTGAC (SEQ ID NO: 73458) (SEQ ID NO:
73459) (SEQ ID NO: 73460) hCV25473653 G/A CTCTAACATCACCGTGTACG
CCTCTAACATCACCGTGTACA GAGCTCTGGGTCAGA- ACTGT (SEQ ID NO: 73461)
(SEQ ID NO: 73462) (SEQ ID NO: 73463) hCV25474627 T/C
GGTACCATGGACTGTACTCACT GTACCATGGACTGTACTCACC AGGTTGGTTCTGG- AGATGAC
(SEQ ID NO: 73464) (SEQ ID NO: 73465) (SEQ ID NO: 73466)
hCV25474661 A/G AGGAACTACGGCGATATCTAA AGGAACTACGGCGATATCTAG
GGGTTTCCCTGGAC- ACAT (SEQ ID NO: 73467) (SEQ ID NO: 73468) (SEQ ID
NO: 73469) hCV25477 A/G GCACAGCACCTTATGTCCA CACAGCACCTTATGTCCG
CCTCTTGAGCATTCATTTGTAA- TT (SEQ ID NO: 73470) (SEQ ID NO: 73471)
(SEQ ID NO: 73472) hCV2548962 C/T CAAATACATCTCCCAGGATC
CAAATACATCTCCCAGGATT GTTTTAATTGCAGTTTG- AATGATAT (SEQ ID NO: 73473)
(SEQ ID NO: 73474) (SEQ ID NO: 73475) hCV25591528 T/C
TCCAAAAGGACCTGACAT TCCAAAAGGACCTGACAC GGCTGCAGAATGGAATTT (SEQ ID
NO: 73476) (SEQ ID NO: 73477) (SEQ ID NO: 73478) hCV25594697 C/T
GCGCGTCTTCCCC CGCGCGTCTTCCCT TCACGTGCTTGACGATTATC (SEQ ID NO:
73479) (SEQ ID NO: 73480) (SEQ ID NO: 73481) hCV25594815 G/A
GCTGGGCCGTCTG AGCTGGGCCGTCTA CACCGCTCATGGACACA (SEQ ID NO: 73482)
(SEQ ID NO: 73483) (SEQ ID NO: 73484) hCV25596020 G/C
AACCCCATTCCCTTGAG AACCCCATTCCCTTGAC AGGAACTCCCTTTGGAGATAT (SEQ ID
NO: 73485) (SEQ ID NO: 73486) (SEQ ID NO: 73487) hCV25598595 A/G
AACAATAGAACATCTTTTCACAA AACAATAGAACATCTTTTCACAG CCAAACACCA-
ACTTGATCATC (SEQ ID NO: 73488) (SEQ ID NO: 73489) (SEQ ID NO:
73490) hCV25602572 G/A GCATTCCAGGTTGAGAG GCATTCCAGGTTGAGAA
CGCACCTGGAGTGATAGAC (SEQ ID NO: 73491) (SEQ ID NO: 73492) (SEQ ID
NO: 73493) hCV25603879 C/T GAAGTCATTCTGCTCTGC ATGGAAGTCATTCTGCTCTGT
TTTCCATCTCCTAACTC- TTTTCTAG (SEQ ID NO: 73494) (SEQ ID NO: 73495)
(SEQ ID NO: 73496) hCV25604100 G/A ACAAGTGTCTGGCTGAGG
CACAAGTGTCTGGCTGAGA TGCCTCAGACACTGAGAAA- TTAT (SEQ ID NO: 73497)
(SEQ ID NO: 73498) (SEQ ID NO: 73499) hCV25605897 G/T
AAGGCAGGATGGGAGTG AAAGGCAGGATGGGAGTT CGTCAAAGCACTAATGTCATG- T (SEQ
ID NO: 73500) (SEQ ID NO: 73501) (SEQ ID NO: 73502) hCV25605906 A/G
CTTCAACTTGGAAAGACATCTTA TCAACTTGGAAAGACATCTTG CTTGAAAGGGTA-
GAAGTAAGACATAT (SEQ ID NO: 73503) (SEQ ID NO: 73504) (SEQ ID NO:
73505)
hCV25607736 T/G GTCGGGAGCTTTCCTGT CTGGGAGCTTTCCTGG
CAGGACAGCACTGGTGTCT (SEQ ID NO: 73506) (SEQ ID NO: 73507) (SEQ ID
NO: 73508) hCV25607748 A/T GCTGTGCTGCTGGTACA GGCTGTGCTGCTGGTACT
GGCATGACCTCTGACATCTC (SEQ ID NO: 73509) (SEQ ID NO: 73510) (SEQ ID
NO: 73511) hCV25608687 C/A GGGTAGTACCAAAAATATTACTTACTT
GGGTAGTACCAAAAATATTACTTACTT CC- AAGTGGAGAAGTGACTAGACA TC (SEQ ID
NO: 73512) TA (SEQ ID NO: 73513) (SEQ ID NO: 73514) hCV25608809 G/A
AGGGAAACCCCAGAGAG AGGGAAACCCCAGAGAA ACAAGTTCATTTGTGAATGTGA- (SEQ ID
NO: 73515) (SEQ ID NO: 73516) (SEQ ID NO: 73517) hCV25610227 C/T
GCAGGTGGCGTATCTGTC GCAGGTGGCGTATCTGTT CGGCCTGGAACCTTAGC (SEQ ID NO:
73518) (SEQ ID NO: 73519) (SEQ ID NO: 73520) hCV25610470 T/C
CACAATCACCATGGTCT ACAATCACCACGGTCC CCTTCTGCATCAGCATCTTC (SEQ ID NO:
73521) (SEQ ID NO: 73522) (SEQ ID NO: 73523) hCV25610773 C/G
ACCCCCCGAAGAACC CCCCCCGAAGAACG GCGTGGAGATCCTGACTC (SEQ ID NO:
73524) (SEQ ID NO: 73525) (SEQ ID NO: 73526) hCV25614016 A/G
AGTGGCCAAGAACACCA TGGCCAAGAACACCG GGTATGAGGCAAAGTTCCTG (SEQ ID NO:
73527) (SEQ ID NO: 73528) (SEQ ID NO: 73529) hCV25616048 C/G
GTTGAAAATGTGAATCAGCAC GTTGAAATGTGAATCAGCAG TGCAGAGCTTCCAAG- TTTT
(SEQ ID NO: 73530) (SEQ ID NO: 73531) (SEQ ID NO: 73533)
hCV25617360 C/T GGGTCTCCCACTCCATAC GGTCTCCCACTCCATAT
GGGCCCATTAACTCACATAC (SEQ ID NO: 73534) (SEQ ID NO: 73535) (SEQ ID
NO: 73536) hCV25617557 T/C GCCTCCCGGAGGACTT GCCTCCCGGAGGACTC
GGAACTAACCATGGCTTCTCTTA (SEQ ID NO: 73537) (SEQ ID NO: 73538) (SEQ
ID NO: 73539) hCV25618313 C/T GGGACCGGATCAGCAC GGGACCGGATCAGCAT
GGCAGTAGGATGAATTAGAAAGTG- (SEQ ID NO: 73540) (SEQ ID NO: 73541)
(SEQ ID NO: 73542) hCV25620145 T/C CACACCAGCAATGATGAAACT
CACCAGCAATGATGAAACC GGGCTAACTCTTTGCA- TGTTC (SEQ ID NO: 73543) (SEQ
ID NO: 73544) (SEQ ID NO: 73545) hCV25623155 G/A CGGAATCGAAACGTGAG
CGGAATCGAAACGTGAA GCACCTCTCGGAGTGTATCT (SEQ ID NO: 73546) (SEQ ID
NO: 73547) (SEQ ID NO: 73548) hCV25623804 T/C TTTCAAGCTGTCTCCTACCAT
TTTCAAGCTGTCTCCTACCAC GGAAGAAGGGAAGG- ACTAAAG (SEQ ID NO: 73549)
(SEQ ID NO: 73550) (SEQ ID NO: 73551) hCV25624076 G/A
AGTTTGAGGATGATGCTATTAG AGTTTGAGGATGATGCTATTAA AGTGGGTTATTG-
TCACTTTTTCTAT (SEQ ID NO: 73552) (SEQ ID NO: 73553) (SEQ ID NO:
73554) hCV25630686 C/T AGGTTGTACCTGTAGCACTAAGAC
TAGGTTGTACCTGTAGCACTAAGAT TGGGCTC- CTCAGAGAAAATAT (SEQ ID NO:
73555) (SEQ ID NO: 73556) (SEQ ID NO: 73557) hCV25631989 C/T
AAGATAAGCCTGTCACTGGTC AAGATAAGCCTGTCACTGGTT CAAGCCAGCCTAAT-
AAACATAA (SEQ ID NO: 73558) (SEQ ID NO: 73559) (SEQ ID NO: 73560)
hCV25632288 A/C TGAGTGTCTCAGTTTCTAGCA GAGTGTCTCAGTTTCTAGCC
CTGAAGAGGACATGT- CAAATATTAC (SEQ ID NO: 73561) (SEQ ID NO: 73562)
(SEQ ID NO: 73563) hCV25636672 C/T GAAACATCAAAATCCTCCAGAC
GAAACATCAAAATTCTCCAGAT TGGGTATTGCAT- TTTTAAGTTTAG (SEQ ID NO:
73564) (SEQ ID NO: 73565) (SEQ ID NO: 73566) hCV25637309 A/T
GGCCACTTTGCCTGAATA GGCCACTTTGCCTGAATT CGAAATGTTCATTTTTAAAG- TCAGA
(SEQ ID NO: 73567) (SEQ ID NO: 73568) (SEQ ID NO: 73569)
hCV25637537 A/G ATGGCCAACTCCTTCA TGGCCAACTCCTTCG GGTCCATGCTCCAGATGA
(SEQ ID NO: 73570) (SEQ ID NO: 73571) (SEQ ID NO: 73572)
hCV25638153 G/C CCTCTCCACACGTTTTG CCTCTCCACAGCGTTTTC
GCAGCGGCCACAGAG (SEQ ID NO: 73573) (SEQ ID NO: 73574) (SEQ ID NO:
73575) hCV25638155 C/T GTGTCTTCCCCCACCAC GTGTCTTCCCCCACCAT
TGCGGCAGCAACTGA (SEQ ID NO: 73576) (SEQ ID NO: 73577) (SEQ ID NO:
73578) hCV25640926 T/C GCCCAGAGACAGGAAAAT GCCCAGAGACAGGAAAAC
GCCTGCCCTCTGTTCA (SEQ ID NO: 73579) (SEQ ID NO: 73580) (SEQ ID NO:
73581) hCV25646246 C/T ATCTACACCATTGCACAC CATCTACACCATTGCACAT
GCCTCCTCCCTTTTCAGT (SEQ ID NO: 73581) (SEQ ID NO: 73582) (SEQ ID
NO: 73583) hCV25651174 T/C CGCTGCAGGGTCAT CGCTGCAGGGTCAC
CCTCCCCGCAGAGAATTA (SEQ ID NO: 73584) (SEQ ID NO: 73585) (SEQ ID
NO: 73586) hCV25651593 G/T TGTCCTGGTTCTGCTCAG ATGTCCTGGTTCTGCTCAT
CTGGTCTCCTTCGTTCAGA- (SEQ ID NO: 73587) (SEQ ID NO: 73588) (SEQ ID
NO: 73589) hCV25652706 C/G GGTGTTCCTGGCTTCC GGTGTTCCTGGCTTCG
GGAGAACCTAACAAGGATTTTACT- A (SEQ ID NO: 73590) (SEQ ID NO: 73591)
(SEQ ID NO: 73592) hCV25653599 C/T AAGAATTTGAACTTACTGATGAGAC
TAAGAATTTGAACTTACTGATGAGAT ATGTG- GAGTTTGATTTCCTTATTA (SEQ ID NO:
73593) (SEQ ID NO: 73594) (SEQ ID NO: 73595) hCV25759173 T/G
AGTCAAAATGGACACAATCTTCT CAAAATGGACACAATCTTCG TGAGATAAGGAAA-
CCTCTTGATAA (SEQ ID NO: 73596) (SEQ ID NO: 73597) (SEQ ID NO:
73598) hCV25771227 G/C CCTGGATGAGGAATCCTACTG CCTGGATGAGGAATCCTACTC
GCTGCCGTACCTGT- TGTAG (SEQ ID NO: 73599) (SEQ ID NO: 73600) (SEQ ID
NO: 73601) hCV25772464 C/T CCCACTGTTGCCATGAC CCCACTGTTGCATGAT
CCTTCCGGCTCCATAAG (SEQ ID NO: 73602) (SEQ ID NO: 73603) (SEQ ID NO:
73604) hCV25922320 T/C CTCGCAGCGGTCAGT TCGCAGCGGTCAGC
GCTGGCGGGAATTTCT (SEQ ID NO: 73605) (SEQ ID NO: 73606) (SEQ ID NO:
73607) hCV25924149 G/C GCAGCGACCATGAG GCAGCGACCATGAC
TCCTCTGCCTCCACTCTG (SEQ ID NO: 73608) (SEQ ID NO: 73609) (SEQ ID
NO: 73610) hCV25925506 G/T GTGTAGTGGTGGAGAGTGG
TGTGATGATGGTGGAGAGTGT ACTGAGCTTGATTTTC- TCTTTTAAT (SEQ ID NO:
73611) (SEQ ID NO: 73612) (SEQ ID NO: 73613) hCV25928236 G/C
GATGGTGAAGTCCTTTATCTCTG GATGGTGAAGTCCTTTATCTCTC AGCACAATTT-
GCAATCACAC (SEQ ID NO: 73614) (SEQ ID NO: 73615) (SEQ ID NO: 73616)
hCV25928842 A/T CACGACGATCCCTTCTGA CACGACGATCCCTTCTGT
GGGCGCAGCTGTCTG (SEQ ID NO: 73617) (SEQ ID NO: 73618) (SEQ ID NO:
73619) hCV25930271 G/A GAATCTCATGTTCAGGAAATG CGAATCTCATGTTCAGGAAATA
GCCATGGCCCATA- AAAC (SEQ ID NO: 73620) (SEQ ID NO: 73621) (SEQ ID
NO: 73622) hCV25931060 C/T CCCAACCCATTGCTCC GCCCAACCCATTGCTCT
TGTCAGCACAGAATCCACTAC (SEQ ID NO: 73623) (SEQ ID NO: 73624) (SEQ ID
NO: 73625) hCV25931248 A/G CTACTTTATCACCTGGCATCA CTTTATCACCTGGCATCG
CACCTCAAGTCTGTGAA- GTACTTAC (SEQ ID NO: 73626) (SEQ ID NO: 73627)
(SEQ ID NO: 73628) hCV25933600 T/C GAGGCAGTGACTAAGGT
AGGCAGTGACTAAGGC GCATGTCAGAATCCTCAATCTC (SEQ ID NO: 73629) (SEQ ID
NO: 73630) (SEQ ID NO: 73631) hCV25941408 G/T CATGGAGTCAACTCTTGAGG
GCATGGAGTCAACTCTTGAGT GGCTGTGCTTTGTCT- GATCT (SEQ ID NO: 73632)
(SEQ ID NO: 73633) (SEQ ID NO: 73634) hCV25944011 C/A
GTATGACCTGTACTTCTGGAGAC GTATGACCTGTACTTCTGGAGAA AGGCCCCCTC-
TCAATACT (SEQ ID NO: 73635) (SEQ ID NO: 73636) (SEQ ID NO: 73637)
hCV26000635 G/T TGCTGGAGCAATTGAGAG CTGCTGGAGCAATTGAGAT
TCTTCCCCTCGTTTCTTTC- (SEQ ID NO: 73638) (SEQ ID NO: 73639) (SEQ ID
NO: 73640) hCV2603660 G/A GGTCATGGCCTTAGAGACTG GGTCATGGCCTTAGAGACTA
GGCCGACCATAGAGATG- AG (SEQ ID NO: 73641) (SEQ ID NO: 73642) (SEQ ID
NO: 73643) hCV2620926 A/G CCTAGTCCTGGGCCTGA CCTAGTCCTGGGCCTGG
CGCCACCTGCTGAAACA (SEQ ID NO: 73644) (SEQ ID NO: 73645) (SEQ ID NO:
73646) hCV2630153 C/G AGATGCAACAGAGAATTTTCTC GATGCAACAGAGAATTTTCTG
CTCTGTTCTTTCAA- TTCTTTAGATG (SEQ ID NO: 73647) (SEQ ID NO: 73648)
(SEQ ID NO: 73649) hCV2676035 A/G AATACACAGTCTTGTTTTAGAATTTTA
AATACACAGTCTTGTTTTAGAATTTTA AAA- TGAGGATGTCTACACAGCTATAT A (SEQ ID
NO: 73650) G (SEQ ID NO: 73651) (SEQ ID NO: 73652) hCV2682687 A/C
AGGAGCTGGAGGAGAA AGGAGCTGGAGGAGAC GAGACCTCAACTTTGTTTAGA (SEQ ID NO:
73653) (SEQ ID NO: 73654) (SEQ ID NO: 73655) hCV2716008 G/C
CTGTTGAAGAAAGCGTACCTAG CTGTTGAAGAAAGCGTACCTAC TGCAACAGTTGTT-
TTTATGA (SEQ ID NO: 73656) (SEQ ID NO: 73657) (SEQ ID NO: 73658)
hCV2741051 C/T GCAGCCAGTTTCTCCC TGCAGCCAGTTTCTCCT
CATGAAATGCTTCCAGGTATT (SEQ ID NO: 73659) (SEQ ID NO: 73660) (SEQ ID
NO: 73661) hCV2741083 G/A GTTCCAACCAGAAGAGAATG
GGTTCCAACCAGAAGAGAATA CTTGCCCCCAACAGTT- AG (SEQ ID NO: 73662) (SEQ
ID NO: 73663) (SEQ ID NO: 73664) hCV2741104 C/T
CGGTTTACCTTGACATTATTC CGGTTTACCTTGACATTATTTT GACTGTTGCCCCTT-
ATCTATGT (SEQ ID NO: 73665) (SEQ ID NO: 73666) (SEQ ID NO: 73667)
hCV277090 A/G CTGAAACTTCCGTGGTAGA GAAACTTCCGTGGTAGG
GATCAGTCCCTGGTTCTGAA (SEQ ID NO: 73668) (SEQ ID NO: 73669) (SEQ ID
NO: 73670) hCV2784540 C/G ACCTTCAAAGCCTTCAGATC CCTTCAAAGCCTTCAGATG
GTGGTTTTGGCTTGATTT- TATAT (SEQ ID NO: 73671) (SEQ ID NO: 73672)
(SEQ ID NO: 73673) hCV2838900 T/C CTGATTCTGCCCCTTTTT
CTGATTCTGCCCCTTTTC TACATGGAAGCCTGAGACTTA- C (SEQ ID NO: 73674) (SEQ
ID NO: 73675) (SEQ ID NO: 73676) hCV2838905 C/A
TTCTCCAGAAACTTGACCATC CTTCTCCAGAAACTTGACCATA GAAGATGATGGTAA-
ACCTGAGTTAT (SEQ ID NO: 73677) (SEQ ID NO: 73678) (SEQ ID NO:
73679) hCV2908485 T/C CAAAGCCATGGTCTTTCAT CAAAGCCATGGTCTTTCAC
CCGCTCTTCGGTTCTACTC- (SEQ ID NO: 73680) (SEQ ID NO: 73681) (SEQ ID
NO: 73682) hCV2932115 C/T TGGACGTGGGCTTTTTC TGGACGTGGGCTTTTTT
GCTGCAGCCCTTTTTCTC (SEQ ID NO: 73683) (SEQ ID NO: 73684) (SEQ ID
NO: 73685) hCV2965593 A/G CAGCAAGTGGAGTTTCCA AGCAAGTGGAGTTTCCG
GAGGGCAGTGCCAGTG (SEQ ID NO: 73686) (SEQ ID NO: 73687) (SEQ ID NO:
73688) hCV2972952 G/C TCACACTCTTCCATATTGTCTG
GTCACACTCTTCCATATTGTCTC AACAATCCTCAC- ACACATCTCTCTT (SEQ ID NO:
73689) (SEQ ID NO: 73690) (SEQ ID NO: 73691) hCV2983036 C/G
GCTGACTTTTTTGCTCTTTC GCTGACTTTTTTGCTCTTTG GCCATTTTCCACAATAA- ATATTT
(SEQ ID NO: 73692) (SEQ ID NO: 73693) (SEQ ID NO: 73694) hCV2983037
C/A TGAACTTTTTCTCAGATTCAGTAAC TGAACTTTTTCTCAGATTCAGTAAA GACCACA-
GCATCAAAGAATGT (SEQ ID NO: 73695) (SEQ ID NO: 73696) (SEQ ID NO:
73697) hCV3011239 G/A GTTCTGAGCCACTAAGCG GGTTCTGAGCCACTAAGCA
TCCCCACTTTCTCATTCTTC- T (SEQ ID NO: 73698) (SEQ ID NO: 73699) (SEQ
ID NO: 73700) hCV3029386 G/T AGAATTGTGTCCAAAGAAGTTG
AAGAATTGTGTCCAAAGAAGTTT AACTGGTATAAT- TTGAATCACATAAAT (SEQ ID NO:
73701) (SEQ ID NO: 73702) (SEQ ID NO: 73703) hCV3035766 T/G
TGCTCCTGACCGCAGT GCTCCTGACCGCAGG GGGAGGTCAGCTTTTACAAA (SEQ ID NO:
73704) (SEQ ID NO: 73705) (SEQ ID NO: 73706) hCV3036180 T/G
GGGAATGCGGATGGT GGAATGCGGATGGG CCAGGCGTCGGAACT (SEQ ID NO: 73707)
(SEQ ID NO: 73708) (SEQ ID NO: 73709) hCV3036181 C/G
TCCGAGCCCACATCC TCCGAGCCCACATCG CATGGAGGAGCTGAGAACAC (SEQ ID NO:
73710) (SEQ ID NO: 73711) (SEQ ID NO: 73712) hCV3046056 A/G
CACCTCCTCATCCCACA ACCTCCTCATCCCACG CCTCCCCCTCCACAAG (SEQ ID NO:
73713) (SEQ ID NO: 73714) (SEQ ID NO: 73715) hCV305149 T/C
TGAATGGCATCCACAAAAT TGAATGGCATCCACAAAAC CCAAAAGCTAAAAAGCACAT-
CT
(SEQ ID NO: 73716) (SEQ ID NO: 73717) (SEQ ID NO: 73718) hCV3068164
C/T GCATCACCTGCATCCTC GCATCACCTGCATCCTT TGGATTGGTTGCTGTTCA (SEQ ID
NO: 73719) (SEQ ID NO: 73720) (SEQ ID NO: 73721) hCV3068176 T/C
TACCACAGCTTGCTCACAT TACCACAGCTCACAC TTTCCCCCATTTTTCAGTT (SEQ ID NO:
73722) (SEQ ID NO: 73723) (SEQ ID NO: 73724) hCV3084793 C/T
CCCGGCTGGGCGCGGACATGGAGGACG CCCGGCTGGGCGCGGACATGGAGGACG CAG-
CTTGCGCAGGTG TTC (SEQ ID NO: 73725) TTT (SEQ ID NO: 73726) (SEQ ID
NO: 73727) hCV3131029 A/G CAAATAGATGGAAACTACTCCA
AAATAGATGGAAACTACTCCG GGCAGCCCTATGGT- TTCT (SEQ ID NO: 73728) (SEQ
ID NO: 73729) (SEQ ID NO: 73730) hCV3135085 C/A CTGGAAATGGTTATGGGC
TACTGGAAATGGTTATGGGA TTTATAGGCGTGAAACTAA- TTCTC (SEQ ID NO: 73731)
(SEQ ID NO: 73732) (SEQ ID NO: 73733) hCV3188386 C/T CGCTGCGCGTGTTC
CGCTGCGCGTGTTT GGAGGAAGGGAAAGGTACAG (SEQ ID NO: 73734) (SEQ ID NO:
73735) (SEQ ID NO: 73736) hCV3188814 G/A GCAGATCGAGTTCCG
GGCAGATCGAGTTCCA CCTAAACTCCATGAAGAAGACATT (SEQ ID NO: 73737) (SEQ
ID NO: 73738) (SEQ ID NO: 73739) hCV3210838 C/T
CTGCATTATTTCTATGACGC TTCTGCATTATTTCTATGACGT CAAAAAATGCCAACA-
GTTTAGA (SEQ ID NO: 73740) (SEQ ID NO: 73741) (SEQ ID NO: 73742)
hCV3212009 T/C GTTCTCCCCTTTCAGTGTCT TCTCCCCTTTCAGTGTCC
TGTCGGTGACTGTTCTGTT- AA (SEQ ID NO: 73743) (SEQ ID NO: 73744) (SEQ
ID NO: 73745) hCV3215915 G/T GCATTCCCGAGGGG TGCATTCCCGAGGGT
TTTCCTTCAGGAGTTGATCTCTA (SEQ ID NO: 73746) (SEQ ID NO: 73747) (SEQ
ID NO: 73748) hCV3216553 A/G GCCATACACCTCTTTCAGGA
CCATACACCTCTTTCAGGG CCAGGAGGCATGTTGATA- AG (SEQ ID NO: 73749) (SEQ
ID NO: 73750) (SEQ ID NO: 73751) hCV3216558 C/T
AGTTCCTGACCTTCACATACC AAGTTCCTGACCTTCACATACT CTATGTCAGCATTT-
GCATCTAA (SEQ ID NO: 73752) (SEQ ID NO: 73753) (SEQ ID NO: 73754)
hCV3254661 G/A GCGTGAGGGTGAGCG GCGTGAGGGTGAGCA
GCTAAGGTGACCCAAACTCTTAC (SEQ ID NO: 73755) (SEQ ID NO: 73756) (SEQ
ID NO: 73757) hCV3259537 G/A ACAGCACAGACTTCACCG GACAGCACAGACTTCACCA
GCCATTCCCCACAGTG (SEQ ID NO: 73758) (SEQ ID NO: 73759) (SEQ ID NO:
73760) hCV342590 C/T AGACAAATTCTCTCATGTCCAC AGACAAATTCTCTCATGTCCAT
TGTTTTTCCAGGAA- AAAGATATTC (SEQ ID NO: 73761) (SEQ ID NO: 73762)
(SEQ ID NO: 73763) hCV370782 C/T TTCTACCCAGGTACTTATCATCC
CTTTCTACCCAGGTACTTATCATCT GGATTCACTG- TGAAAGAACAGTAT (SEQ ID NO:
73764) (SEQ ID NO: 73765) (SEQ ID NO: 73765) hCV43568 T/C
GAGGTCTTGAAATACAGGGATT GAGGTCTTGAAATACAGGGATC TCTTGAGAGGTGTGA-
TCATAACTT (SEQ ID NO: 73766) (SEQ ID NO: 73767) (SEQ ID NO: 73768)
hCV517658 T/C AATGGCCTTGGACTTGAT AATGGCCTTGGACTTGAC
CTCTGCCATGCAAAACAC (SEQ ID NO: 73770) (SEQ ID NO: 73771) (SEQ ID
NO: 73772) hCV529706 G/C GCGAGGACGAAGGGG GCGAGGACGAAGGGC
GGAGGATGAATGGACAGACAA (SEQ ID NO: 73773) (SEQ ID NO: 73774) (SEQ ID
NO: 73775) hCV529710 G/C CCGACCCGAACTAAAGG CCGACCCGAACTAAAGC
CGCGTTCCCCATGTC (SEQ ID NO: 73776) (SEQ ID NO: 73777) (SEQ ID NO:
73778) hCV5687 T/C CCCTCAGTGTGACTGAGAT CCCTCAGTGTGACTGAGAC
CCAGGCATTTCCCATACAG (SEQ ID NO: 73779) (SEQ ID NO: 73780) (SEQ ID
NO: 73781) hCV596331 T/C TCCACATCAGGAAAAACAGT CCACATCAGGAAAAACAGC
CATTTGCCAATGAGAAATA- TCA (SEQ ID NO: 73782) (SEQ ID NO: 73783) (SEQ
ID NO: 73784) hCV598677 G/T CCAAGCTGAAAGGCAAG CCAAGCTGAAAGGCAAT
CAGCCAGGGTGGAGAGT (SEQ ID NO: 73785) (SEQ ID NO: 73786) (SEQ ID NO:
73787) hCV7441704 A/G CCAACCGAGATCAGATTGA CAACCGAGATCAGATTGG
TGATGCTGATTGTGGATGAT- A (SEQ ID NO: 73788) (SEQ ID NO: 73789) (SEQ
ID NO: 73790) hCV7449215 T/C CCCAGGCCCAGTTCAT CCCAGGCCCAGTTCAC
CATGTCTGGGCTGGAGAGTA (SEQ ID NO: 73791) (SEQ ID NO: 73792) (SEQ ID
NO: 73793) hCV7482175 A/G TGCCCAGGGCTCTGATA GCCCAGGGCTCTGATG
CCATCGCATGCTCAATACA (SEQ ID NO: 73794) (SEQ ID NO: 73795) (SEQ ID
NO: 73796) hCV7482175 A/G TGCCCAGGGCTCTGATA GCCCAGGGCTCTGATG
CCATCGCATGCTCAATACA (SEQ ID NO: 73797) (SEQ ID NO: 73798) (SEQ ID
NO: 73799) hCV7489257 A/G TCCCAAAACCTGGAGACTA CCCAAAACCTGGAGACTG
TCATGGCATCTTCCTTCAA (SEQ ID NO: 73800) (SEQ ID NO: 73801) (SEQ ID
NO: 73802) hCV7490119 G/C GCCTTGGGGCACATG GCCTTGGGGCACATC
GGAATTTATGGCAGTTTTAAACAT (SEQ ID NO: 73803) (SEQ ID NO: 73804) (SEQ
ID NO: 73805) hCV7490135 G/A GCAGTCCTGAACAAAGTAGATG
CGCAGTCCTGAACAAAGTAGATA CGTGCATGTTTT- GAAAAATGTA (SEQ ID NO: 73806)
(SEQ ID NO: 73807) (SEQ ID NO: 73808) hCV7490146 C/T
GCGCTCTGTTCCTTTGTATC GCGCTCTGTTCCTTTGTATT CCTTTCTCCTGCAGAAA- TAAGA
(SEQ ID NO: 73809) (SEQ ID NO: 73810) (SEQ ID NO: 73811) hCV7497135
A/G CACTGGAGAATGCACA CACTGGAGAATGCACG GTGTTCCTGGCTCACAGAA (SEQ ID
NO: 73812) (SEQ ID NO: 73813) (SEQ ID NO: 73814) hCV7499900 T/C
CACACCAGCAATGATGAAACT CACCAGCAATGATGAAACC GGCGGGTTCCAGACAA (SEQ ID
NO: 73815) (SEQ ID NO: 73816) (SEQ ID NO: 73817) hCV7514870 A/C
CATCTTGCCCACAGCAA CATCTTGCCCACAGCAC GAAGTGGGCACTGAACAACT (SEQ ID
NO: 73818) (SEQ ID NO: 73819) (SEQ ID NO: 73820) hCV7538986 G/A
CAGGGATGTTATTATGGTCG TCAGGGATGTTATTATGGTCA CTGTCTGGGTGGGAAT- GTA
(SEQ ID NO: 73821) (SEQ ID NO: 73822) (SEQ ID NO: 73823) hCV7559757
T/G GCATTAACTGCTCCTGGGT CATTAACTGCTCCTGGGG CAACACACGAGCTACAAACT-
ACA (SEQ ID NO: 73824) (SEQ ID NO: 73825) (SEQ ID NO: 73826)
hCV7565899 C/T AAGATATCAATGTTTCTGTCTGTTC AAGATATCAATGTTTCTGTCTGTTT
ATCACTG- GTTCCTTCAACTGT (SEQ ID NO: 73827) (SEQ ID NO: 73828) (SEQ
ID NO: 73829) hCV7574719 G/A CGCTCAGTAACCTGCG GCGCTCAGTAACCTGCA
GGTCTGGGCCTTTCATAAG (SEQ ID NO: 73830) (SEQ ID NO: 73831) (SEQ ID
NO: 73832) hCV7591528 G/A CTCATAGCTTGACCTTCGAG CTCATAGCTTGACCTTCGAA
ACTCCTCATCAGTCACA- GACAC (SEQ ID NO: 73833) (SEQ ID NO: 73834) (SEQ
ID NO: 73835) hCV7615375 C/T CTTCGTCGCAATGGC TTCTTCGTCGCAATGGT
GCAATTTCTGCACAGAAATATT (SEQ ID NO: 73836) (SEQ ID NO: 73837) (SEQ
ID NO: 73838) hCV7615376 G/A GATGAGATCAACACAATCTTCAG
GATGAGATCAACACAATCTTCAA CCTGAAGCTCG- TTTTGAATAA (SEQ ID NO: 73839)
(SEQ ID NO: 73840) (SEQ ID NO: 73841) hCV761961 G/A
CACAGTCPAAGAATCAAGCG TCACAGTCAAAGAATCAAGCA CCGTTTGAATTTTCCAA- TAAG
(SEQ ID NO: 73842) (SEQ ID NO: 73843) (SEQ ID NO: 73844) hCV7798230
G/G GAGCGAGGGCTCAGG GAGCGAGGGCTCAGC CCTCCCTGGAGAATACTGTG (SEQ ID
NO: 73845) (SEQ ID NO: 73846) (SEQ ID NO: 73847) hCV789270 G/C
CCTTTCCCCAAACAGC CCTTTCCCCAAACAGG CATCTCAGCCTCCTTACCA (SEQ ID NO:
73848) (SEQ ID NO: 73849) (SEQ ID NO: 73850) hCV7900503 C/T
CGTCTCCAGGAAAATCATAAC CGTCTCCAGGAAAATCATAAT TGAGTTATTGCTACT-
TCAGAATCAT (SEQ ID NO: 73851) (SEQ ID NO: 73852) (SEQ ID NO: 73853)
hCV790057 A/G AGCAGCTCCGAGTCCA AGCAGCTCCGAGTCCG GGCCCACAAGGTGAAAT
(SEQ ID NO: 73854) (SEQ ID NO: 73855) (SEQ ID NO: 73856) hCV795441
G/C TGTGGGCCAGGACG CTGTGGGCCAGGACC ACCCACCAGGACCTAAAAG (SEQ ID NO:
73857) (SEQ ID NO: 73858) (SEQ ID NO: 73859) hCV795442 G/A
CATTCAATGCAATACGTCG CCATTCAATGCAATACGTCA TGGTCCTGGCCTGAAC (SEQ ID
NO: 73860) (SEQ ID NO: 73861) (SEQ ID NO: 73862) hCV8400671 A/G
TTGTTAACATATACTTACTGGAGA TGTTAACATATACTTACTGGAGG TGCCTCTTCT-
TTATTTATGTC (SEQ ID NO: 73863) (SEQ ID NO: 73854) (SEQ ID NO:
73865) hCV8692704 C/A CTGAGGACCCTTGGAGAC CTGAGGACCCTTGGAGAA
GCTCACCAGCCCTGAAG (SEQ ID NO: 73866) (SEQ ID NO: 73867) (SEQ ID NO:
73868) hCV8695674 A/C ACCGGCACAAGGAGAA ACCGGCACAAGGAGAC
TGCCCTTGTCACTTTCTGTAT (SEQ ID NO: 73869) (SEQ ID NO: 73870) (SEQ ID
NO: 73871) hCV8705506 G/C CCACTTCGGGTTCCTC CCACTTCGGGTTCCTG
CCCTGGCTTCAACATGA (SEQ ID NO: 73872) (SEQ ID NO: 73873) (SEQ ID NO:
73874) hCV8708464 A/G CCCTCTCCAGCGGGA CCTCTCCAGCGGGG
CCAAAGCAGGGTTCACTACC (SEQ ID NO: 73875) (SEQ ID NO: 73876) (SEQ ID
NO: 73877) hCV8709053 G/A GCCCAGATACCCCAAAG GCCCAGATACCCCAAAA
GCGGCTTCAGCAGATC (SEQ ID NO: 73878) (SEQ ID NO: 73879) (SEQ ID NO:
73880) hCV8718197 A/G CCTCTGAGGCCTGAGAAA CCTCTGAGGCCTGAGAAG
GTCCTGATTCCTCATTTCTTT- C (SEQ ID NO: 73881) (SEQ ID NO: 73882) (SEQ
ID NO: 73883) hCV8722981 G/A GCGCTGGTTTGGAGG GCGCTGGTTTGGAGA
TGGCACAGGCAGTATTAAGTAG (SEQ ID NO: 73884) (SEQ ID NO: 73885) (SEQ
ID NO: 73886) hCV8726331 A/G TGGTCTGTTCCCTGGACA GGTCTGTTCCCTGGACG
TGCGGTCACACTGACTGAG (SEQ ID NO: 73887) (SEQ ID NO: 73888) (SEQ ID
NO: 73889) hCV8774272 T/C CCGCTTCCGAGCAGTT CCGCTTCCGAGCAGTC
CGTCAACATCCTCTTTGAAGAA (SEQ ID NO: 73890) (SEQ ID NO: 73891) (SEQ
ID NO: 73892) hCV8784787 A/C ACTTCTGGGGCTTAGGAA ACTTCTGGGGCTTAGGAC
TTCACCGGGAACTCTTGT (SEQ ID NO: 73893) (SEQ ID NO: 73894) (SEQ ID
NO: 73895) hCV8827241 G/C TCAAGAGGACAGTGATGGTG TCAAGAGGACAGTGATGGTC
TGGTTAGAATCTGTGAA- GGAACTA (SEQ ID NO: 73896) (SEQ ID NO: 73897)
(SEQ ID NO: 73898) hCV8851065 G/C CCCCGCAGAGAATTACC
CCCCGCAGAGAATTACG ACGTCGCTGTCGAAGC (SEQ ID NO: 73899) (SEQ ID NO:
73900) (SEQ ID NO: 73901) hCV8851074 T/G TGGCTGTTCCAGTACTCCT
GGCTGTTCCAGTACTCGG TTCCTGGAGAGATACATCTA- CAAC (SEQ ID NO: 73902)
(SEQ ID NO: 73903) (SEQ ID NO: 73904) hCV8851080 T/C GGCACTGCCCGCTT
GGCAGTGCCCGCTC CGCTTCCTGGAGAGATACATC (SEQ ID NO: 73905) (SEQ ID NO:
73906) (SEQ ID NO: 73907) hCV8851084 A/G CAGTGCCGGACAGGA
CAGTGCCGGACAGGG CCGCCCGGCACTAAG (SEQ ID NO: 73908) (SEQ ID NO:
73909) (SEQ ID NO: 73910) hCV8851085 T/C GCTCGTAGTTGTGTCTGGAT
GCTCGTAGTTGTGTCTGCAC CGCTTCCTGGAGAGATA- CAT (SEQ ID NO: 73911) (SEQ
ID NO: 73912) (SEQ ID NO: 73913) hCV8884725 T/C GCATTTTCACTCCGAAGTT
GCATTTTCACTGCGAAGTC CATGACTAGCTCTCATTTT- GATTAG (SEQ ID NO: 73914)
(SEQ ID NO: 73915) (SEQ ID NO: 73916) hCV8903097 G/T
GACATGGGGGAGTCAC GACATGGGGGAGTCAT GCATCTCGGGTTCTACTT (SEQ ID NO:
73917) (SEQ ID NO: 73918) (SEQ ID NO: 73919) hCV8919441 G/T
AGATCACTAGGAGGGTCCTC AGATCACTAGGAGGGTCCTT GGTTCTCCTGGATGAAA- TTACTA
(SEQ ID NO: 73920) (SEQ ID NO: 73921) (SEQ ID NO: 73922) hCV8919442
C/T TTCTACCTTGGGTCCCTTAC TTGTACGTTGGGTCCCTTAT TTCCAGCCCATATTCTG- AA
(SEQ ID NO: 73923) (SEQ ID NO: 73924) (SEQ ID NO: 73925) hCV8919444
G/A TGGTGCTGGAGAATTCAG TGGTGCTGGAGAATTCAA GGTGTCTCCCAACTTTATGTG-
(SEQ ID NO: 73926) (SEQ ID NO: 73927) (SEQ ID NO: 73928) hCV8919523
T/C GTGAGGAGGGATTTGAATTAT GTGAGGAGGGATTTGAATTAC AAAAAAGTCTGCACT-
CAATTCTAC (SEQ ID NO: 73929) (SEQ ID NO: 73930) (SEQ ID NO: 73931)
hCV8921130 C/T AGGCCGTCACTGTAC AGGCCGTCAGTGTAT CCATTTCCTCCCAAATACAT
(SEQ ID NO: 73932) (SEQ ID NO: 73933) (SEQ ID NO: 73934) hCV8921288
G/T CCGCAGAGGTGTGGG CGGCAGAGGTGTGGT CATTTTGCGGTGGAAATG (SEQ ID NO:
73935) (SEQ ID NO: 73936) (SEQ ID NO: 73937)
hCV8928919 G/A GGTTCCCGAGGAAAGAAG GGTTCCCGAGGAAAGAAA
CGTCCTCGCTCACCTTAAA (SEQ ID NO: 73938) (SEQ ID NO: 73939) (SEQ ID
NO: 73940) hCV8932279 A/G GAGTGGCCCTATCAAATGTTA GTGGCCCTATCAAATGTTG
TTTGTTGTGCCTGATGA- TGTA (SEQ ID NO: 73941) (SEQ ID NO: 73942) (SEQ
ID NO: 73943) hCV8933098 P/G TGCAAATACTGCCAACCA GCAAATACTGCCAACCG
GAGGCAGTGUGATTTAAGAAGA- (SEQ ID NO: 73944) (SEQ ID NO: 73945) (SEQ
ID NO: 73946) hCV8934009 C/G CCTGGTTTCACTGTAGTCACTC
CCTGGTTTCACTGTAGTCACTG TGGCCACAGGAAT- CTGTC (SEQ ID NO: 73947) (SEQ
ID NO: 73948) (SEQ ID NO: 73949) hCV8952817 G/C
CGCATCCAGAACATTCTATG CGCATCCAGAACATTCTATC GCAGCTTCCCATCATAC- ACT
(SEQ ID NO: 73950) (SEQ ID NO: 73951) (SEQ ID NO: 73952) hCV699804
T/G GTGCATTCTGCTTTTAACTCAT TGCATTCTGCTTTTAACTCAG GCAATTGCCTGCTCA-
TTAGT (SEQ ID NO: 73953) (SEQ ID NO: 73954) (SEQ ID NO: 73955)
hCV901792 C/G ATGACAAGTCTCTGAATAAGAAGTC TGACAAGTCTCTGAATAAGAAGTG
TGAATGCTT- TGATCACATGAGT (SEQ ID NO: 73956) (SEQ ID NO: 73957) (SEQ
ID NO: 73958) hCV9458082 C/T ACAGTCAGGTGGATCTCC CACAGTCAGGTGGATCTCT
CACATTCCTGGAGGTGCTAG- (SEQ ID NO: 73959) (SEQ ID NO: 73960) (SEQ ID
NO: 73961) hCV9458936 A/G CCCCAACCCAAGAGAAA CCCCAACCCAAGAGAAG
TCCAGGGCTGCTTACTTC (SEQ ID NO: 73962) (SEQ ID NO: 73963) (SEQ ID
NO: 73964) hCV9482394 G/A CGCGCGCTAACCG CGCGCGCTAACCA
CGTCCGTCATGGATCAGA (SEQ ID NO: 73965) (SEQ ID NO: 73966) (SEQ ID
NO: 73967) hCV9485713 T/C GCCCAGAGACAGGAAAAT GCCCAGAGACAGGAAAAC
GCCTGCCCTCTGTTCA (SEQ ID NO: 73968) (SEQ ID NO: 73969) (SEQ ID NO:
73970) hCV9494470 C/A AGGGATCCGCAAAGC CAGGGATCCGCAATAGA
TCTTTCTGCCAGGTACATCA (SEQ ID NO: 73971) (SEQ ID NO: 73972) (SEQ ID
NO: 73973) hCV9506149 T/A CTGCTGGCCGTCCT TGCTGGCCGTCCA
ACTCACGCTTGCTTTGACT (SEQ ID NO: 73974) (SEQ ID NO: 73975) (SEQ ID
NO: 73976) hCV9514434 T/C CCTTGAAAGATCTCCCTCTTT
CCTTGAAAGATCTCCCTCTTC CGTAGCCCCAAAGGA- TCT (SEQ ID NO: 73977) (SEQ
ID NO: 73978) (SEQ ID NO: 73979) hCV9546471 A/C
CTCAGGAAGCTAAAAGGTGA TCAGGAAGCTAAAAGGTGC CCTAATATCCCCTCCAGA- ACTAT
(SEQ ID NO: 73980) (SEQ ID NO: 73981) (SEQ ID NO: 73982) hCV9596962
G/A CAGCGGAAGCCAAGG CAGCGGAAGCCAAGA CCGGGAGATGAAGAAGAGA (SEQ ID NO:
73983) (SEQ ID NO: 73984) (SEQ ID NO: 73985) hCV9604851 T/C
CCGCCTTGCAGATGAT CCGCCTTGCAGATGAC GGAGCTGGCCATTAGAATC (SEQ ID NO:
73986) (SEQ ID NO: 73987) (SEQ ID NO: 73988) hCV9615318 C/T
GCTCAGATCTGAACCCTAACTC GCTCAGATCTGAACCCTAACTT TCACCCCCTCCTG- ACC
(SEQ ID NO: 73989) (SEQ ID NO: 73990) (SEQ ID NO: 73991) hCV9689262
C/T GGTTGTGCAGAGCAGTTAAC GGTTGTGCAGAGCAGTTAAT TGGCTGTGTTTTGAAAA-
ACTA (SEQ ID NO: 73992) (SEQ ID NO: 73993) (SEQ ID NO: 73994)
hCV216064 A/G TGCCCAGGGCTCTGATA GCCCAGGGCTCTGATG
CCATCGCATGCTCAATACA (SEQ ID NO: 73995) (SEQ ID NO: 73996) (SEQ ID
NO: 73997)
TABLE-US-00007 TABLE 6 Case Control Marker Study Stratification
Strata Status Allele1 Allele1 frq Allele1 frq Allelic p- value Dom
p- value Rec p- value OR OR 95% CI L OR 95% CI U hCV25632288 S0028
SMOKE N MI_YOUNGOLD_noASD C 0.00 0.06 0.0262 0.07 0 1.- 68
hCV25632288 S0012(F) no ALL MI_noASD C 0.02 0.05 0.0056 0.42 0.22
0.77 hCV16196014 S0012 HTN N MI_noMI G 0.07 0.04 0.03 0.02 0.49
1.76 1.09 2.85 hCV16196014 S0012 SMOKE Y MI_noASD G 0.06 0.05 1.00
0.74 0.05 1.01 0.49 2.- 06 hCV16196014 S0028 no ALL MI_noASD G 0.05
0.03 0.04 0.06 0.19 1.91 1.04 3.5- 1 hCV16196014 S0028 SMOKE Y
MI_noMI G 0.05 0.04 0.19 0.32 0.02 1.38 0.88 2.1- 8 hCV8692704
S0028 AGE_LT60 Y MI_noMI A 0.57 0.51 0.0379 1.30 1.01 1.66
hCV8692704 S0028 AGE_LT60 Y MI_noMI A 0.57 0.51 0.0379 1.30 1.02
1.63 hCV8692704 S0012(F) no ALL YoungMI_GT75noASD A 0.57 0.49
0.0293 1.39 1.0- 8 1.78 hCV8692704 S0012(M) no ALL LT60MI_GT75noMI
A 0.56 0.47 0.0204 1.41 1.06 - 1.88 hCV25631989 S0028 no ALL
MI_noMI T 0.06 0.09 0.0265 0.68 0.48 0.94 hCV25631989 S0012(M)
AGE_LT60 O MI_LT75noMI T 0.05 0.10 0.0335 0.44 0.2 - 0.93
hCV11506744 S0028 no ALL MI_noMI G 0.40 0.46 0.0033 0.77 0.64 0.91
hCV11506744 S0012(M) no ALL LT60MI_60TO75noMI G 0.39 0.45 0.0497
0.76 0.- 57 0.99 hCV8884725 S0028 no ALL MI_noASD C 0.01 0.03
0.0138 0.41 0.21 0.79 hCV8884725 S0028 no ALL MI_noMI C 0.02 0.03
0.0262 0.51 0.28 0.9 hCV8884725 S0012(F) no ALL YoungMI_GT75noASD C
0.01 0.04 0.0228 0.32 0.1- 2 0.81 hCV25637537 S0012 HTN Y MI_noMI A
0.07 0.05 0.04 0.03 1.53 1.02 2.29 hCV25637537 S0028 no ALL
MI_noASD A 0.06 0.03 0.01 0.01 0.44 2.13 1.18 3.8- 2 hCV25637537
S0028 no ALL MI_noMI A 0.06 0.04 0.02 0.02 0.54 1.56 1.08 2.24-
hCV25637537 S0028 no ALL MI_YOUNGOLD_noASD A 0.05 0.01 0.04 0.03
3.62 1.0- 6 12.32 hCV25637537 S0028 HTN Y MI_noASD A 0.06 0.03 0.06
0.05 0.51 1.95 1.00 3.79- hCV25637537 S0028 HTN Y MI_noMI A 0.06
0.04 0.05 0.04 0.64 1.49 1.01 2.19 hCV8851074 S0012 SMOKE N
MI_noASD A 0.28 0.33 0.21 0.03 0.46 0.79 0.56 1.1- 2 hCV8851074
S0012 SMOKE N MI_noMI A 0.28 0.33 0.06 0.03 0.61 0.78 0.60 1.01-
hCV8851074 S0028 no ALL MI_noASD A 0.33 0.36 0.27 0.03 0.41 0.88
0.70 1.10- hCV8851074 S0028 no ALL MI_noMI A 0.33 0.37 0.05 0.00
0.73 0.84 0.71 1.00 hCV8851074 S0028 SMOKE N MI_noASD A 0.30 0.39
0.05 0.03 0.45 0.66 0.45 0.9- 8 hCV8851074 S0028 SMOKE N MI_noMI A
0.30 0.39 0.02 0.02 0.21 0.66 0.47 0.93- hCV25931248 V0001 no ALL
MI_noMI A 0.16 0.20 0.0202 0.0067 0.9257 0.79 0.6- 5 0.96
hCV25931248 S0012(M) no ALL MI_noMI G 0.83 0.78 0.0062 1.42 1.1
1.82 hCV1403468 V0001 SEX M MI_noMI G 0.13 0.18 0.0075 0.7 0.4 0.8
hCV1403468 S0012(M) no ALL MI_GT75noMI G 0.16 0.22 0.0252 0.7 0.4
0.9 hCV11482579 S0012 SEX M MI_YOUNGOLD_noASD T 0.08 0.13 0.26 0.46
0.04 0.59 - 0.23 1.47 hCV11482579 S0028 SEX M MI_YOUNGOLD_noASD T
0.08 0.09 0.63 0.86 0.05 0.78 - 0.32 1.90 hCV2620926 S0012 no ALL
MI_noASD A 0.10 0.15 0.05 0.02 0.92 0.58 0.35 0.97- hCV2620926
S0012 SMOKE N MI_noMI A 0.08 0.11 0.04 0.04 0.39 0.65 0.43 0.98-
hCV2620926 S0028 SMOKE N MI_noASD A 0.09 0.16 0.03 0.03 0.32 0.52
0.29 0.9- 3 hCV2620926 S0028 HTN Y MI_noASD A 0.12 0.17 0.03 0.03
0.21 0.68 0.48 0.95 hCV8903097 S0028 SEX F MI_YOUNGOLD_noASD T 0.82
0.93 0.0380 0.37 0.21 0.- 63 hCV8903097 S0012(F) no ALL MI_noASD T
0.86 0.90 0.0178 0.65 0.48 0.87 hCV15869253 S0028 SMOKE N MI_noMI T
0.08 0.13 0.0382 0.0276 0.7584 0.60 0.- 37 0.97 hCV15869253
S0012(M) no ALL MI_noMI T 0.11 0.15 0.048 0.74 0.55 0.99
hCV15869253 S0012(M) no ALL MI_LT75noMI T 0.11 0.16 0.009 0.66 0.48
0.9 hCV25931060 S0028 AGE_LT60 Y MI_noMI A 0.23 0.16 0.0097 1.51
1.13 1.99 hCV25931060 S0012(F) AGE_LT60 Y MI_noASD A 0.22 0.16
0.0373 1.49 1.1 2.0- 1 hCV7441704 S0028 no ALL MI_noASD G 0.03 0.06
0.0386 0.57 0.35 0.9 hCV7441704 S0012(F) no ALL YoungMI_GT75noASD G
0.03 0.08 0.0321 0.41 0.1- 9 0.88 hCV25653599 V0001 no ALL MI_noMI
C 0.14 0.11 0.0256 0.0312 0.3216 1.31 1.0- 3 1.67 hCV25653599
S0012(M) no ALL MI_noMI T 0.83 0.87 0.0107 0.70 0.53 0.92
hCV25653599 S0012(M) no ALL MI_LT75noMI T 0.83 0.88 0.0042 0.64
0.46 0.8- 6 hCV1486426 V0001 no ALL MI_noMI A 0.03 0.05 0.0174
0.0174 0.4091 0.63 0.44- 0.91 hCV1486426 S0012(M) no ALL MI_noMI G
0.97 0.95 0.0418 1.69 1.02 2.8 hCV25928842 S0028 SEX M MI_noASD T
0.96 0.99 0.02180 0.32 0.13 0.76 hCV25928842 S0028 SEX M MI_noMI T
0.96 0.99 0.00935 0.30 0.12 0.7 hCV25928842 S0028 SMOKE Y MI_noMI T
0.97 0.99 0.00710 0.28 0.11 0.68 hCV25928842 S0012(M) no ALL
MI_noMI T 0.95 0.98 0.00185 0.43 0.24 0.74 hCV25928842 S0012(M) no
ALL MI_LT75noMI T 0.95 0.99 0.00000 0.15 0.06 0.- 38 hCV25928842
S0012(M) SMOKE Y MI_LT75noMI T 0.97 0.99 0.02132 0.25 0.07 0- .86
hCV25928236 V0001 HTN N MI_noMI C 0.01 0.02 0.0399 0.0331 0.46 0.22
0.96 hCV25928236 S0012(M) no ALL MI_noMI G 1.00 0.98 0.0033 4.51
1.41 14.36 hCV25928236 S0012(M) no ALL MI_LT75noMI G 1.00 0.98
0.0042 4.81 1.46 15.- 74 hCV25933600 S0028 no ALL MI_noASD C 0.69
0.76 0.0113 0.73 0.56 0.92 hCV25933600 S0012(M) no ALL MI_noMI C
0.70 0.74 0.0455 0.80 0.64 0.98 hCV3215915 S0028 SMOKE N MI_noASD T
0.71 0.79 0.0289 0.63 0.44 0.88 hCV3215915 S0012(F) no ALL MI_noASD
T 0.66 0.73 0.0108 0.73 0.58 0.9 hCV25930271 S0028 no ALL MI_noASD
A 0.95 0.99 0.0000 0.17 0.1 0.25 hCV25930271 S0028 SMOKE Y MI_noASD
A 0.95 0.99 0.0045 0.14 0.08 0.22 hCV25930271 S0012(F) SMOKE Y
MI_noASD A 0.96 1.00 0.0052 0.10 0.04 0.21 hCV15853800 S0028 no ALL
MI_YOUNGOLD_noASD G 0.01 0.04 0.0165 0.17 0.04 - 0.64 hCV15853800
S0012(F) AGE_LT60 Y MI_noASD G 0.01 0.03 0.0334 0.28 0.09 0.- 86
hCV2143205 S0012 no ALL MI_YOUNGOLD_noASD A 0.24 0.33 0.06 0.03
0.65 0.64 - 0.41 1.01 hCV2143205 S0012 HTN N MI_YOUNGOLD_noASD A
0.21 0.34 0.06 0.02 0.64 0.51 0- .26 0.99 hCV2143205 S0028 HTN N
MI_YOUNGOLD_noASD A 0.13 0.37 0.03 0.05 0.16 0.24 0- .06 0.92
hCV7798230 S0028 AGE_LT60 Y MI_noASD C 0.70 0.61 0.0171 1.46 1.12
1.89 hCV7798230 S0012(F) no ALL MI_noASD C 0.74 0.68 0.0329 1.31
1.04 1.64 hCV7798230 S0012(F) AGE_LT60 Y MI_noASD C 0.74 0.65
0.0113 1.52 1.15 1.9- 9 hCV1276216 S0012 BMI_GE27 H MI_noMI G 0.28
0.32 0.06 0.03 0.59 0.80 0.64 1- .01 hCV1276216 S0012 SEX M MI_noMI
G 0.31 0.35 0.05 0.06 0.20 0.81 0.66 0.99 hCV1276216 S0028 BMI_GE27
H MI_noMI G 0.31 0.35 0.08 0.04 0.57 0.83 0.68 1- .02 hCV1276216
S0028 SEX M MI_noMI G 0.31 0.35 0.05 0.04 0.36 0.80 0.65 1.00
hCV9506149 S0012 no ALL MI_noMI T 0.28 0.23 0.01 0.02 0.14 1.27
1.05 1.53 hCV9506149 S0028 no ALL MI_noMI T 0.28 0.25 0.09 0.05
0.70 1.17 0.98 1.39 hCV11592758 S0028 no ALL MI_noASD C 0.86 0.81
0.00369 1.42 1.11 1.79 hCV11592758 S0028 no ALL MI_noMI C 0.86 0.82
0.01264 1.34 1.06 1.68 hCV11592758 V0001 no ALL MI_noMI C 0.82 0.79
0.0263 1.26 1.02 1.53 hCV11592758 S0012(M) no ALL MI_noMI C 0.84
0.78 0.00721 1.42 1.1 1.83 hCV11592758 S0012(M) no ALL MI_LT75noMI
C 0.84 0.78 0.00929 1.44 1.1 1.8- 9 hCV2146578 S0028 no ALL MI_noMI
T 0.44 0.39 0.0120 1.26 1.07 1.47 hCV2146578 S0012(F) no ALL
MI_noASD T 0.47 0.40 0.0184 1.32 1.07 1.61 hCV7900503 S0028 SMOKE N
MI_YOUNGOLD_noMI T 0.15 0.30 0.0301 0.42 0.19 0- .86 hCV7900503
S0028 AGE_LT60 O MI_noASD T 0.18 0.28 0.0116 0.57 0.37 0.86
hCV7900503 S0012(M) no ALL MI_noMI T 0.19 0.25 0.0077 0.72 0.56
0.91 hCV8921288 S0012 SEX M MI_YOUNGOLD_noASD G 0.20 0.15 0.20 0.03
0.10 1.40 0- .86 2.26 hCV8921288 S0012 SMOKE N MI_YOUNGOLD_noASD G
0.24 0.16 0.02 0.01 0.87 1.68- 1.08 2.63 hCV8921288 S0028 no ALL
MI_noASD G 0.22 0.17 0.03 0.03 0.46 1.34 1.02 1.77- hCV8921288
S0028 SEX M MI_noASD G 0.22 0.17 0.12 0.02 0.33 1.36 0.93 1.97
hCV16171764 S0012 SEX M MI_noASD T 0.03 0.06 0.06 0.11 0.04 0.47
0.22 1.03- hCV16171764 S0028 SEX M MI_YOUNGOLD_noASD T 0.04 0.07
0.38 0.73 0.05 0.65 - 0.22 1.88 hCV2259750 S0028 no ALL MI_noMI A
0.38 0.34 0.0324 0.0625 0.0934 1.20 1.02- 1.41 hCV2259750 S0012(M)
no ALL LT60MI_GT75noMI A 0.37 0.30 0.040 1.38 1.01 1- .86
hCV2259750 S0012(M) no ALL MI_GT75noMI A 0.37 0.30 0.038 1.36 1.01
1.8 hCV2548962 S0012 no ALL MI_noASD C 0.28 0.36 0.09 0.40 0.02
0.72 0.50 1.05- hCV2548962 S0012 BMI_GE27 L MI_noMI C 0.27 0.33
0.07 0.01 0.93 0.78 0.59 1- .01 hCV2548962 S0028 BMI_GE27 L
MI_noASD C 0.21 0.33 0.01 0.05 0.01 0.55 0.35 - 0.87 hCV2548962
S0028 BMI_GE27 L MI_noMI C 0.21 0.32 0.01 0.02 0.02 0.58 0.39 0-
.84 hCV25598595 S0012 no ALL MI_YOUNGOLD_noASD G 0.12 0.09 0.12
0.05 0.37 1.42- 0.92 2.18 hCV25598595 S0012 SEX F MI_noASD G 0.11
0.06 0.05 0.02 0.18 1.83 1.00 3.35- hCV25598595 S0012 SEX F
MI_YOUNGOLD_noASD G 0.13 0.06 0.02 0.01 0.30 2.30 - 1.17 4.53
hCV25598595 S0012 HTN Y MI_YOUNGOLD_noASD G 0.13 0.07 0.04 0.04
0.37 1.96 - 1.02 3.76 hCV25598595 S0028 no ALL MI_noASD G 0.12 0.08
0.0190 0.0286 0.1107 1.55 1.- 08 2.23 hCV25598595 S0028 no ALL
MI_noMI G 0.12 0.09 0.0018 0.0025 0.0943 1.49 1.1- 7 1.91
hCV25598595 S0028 no ALL MI_YOUNGOLD_noASD G 0.12 0.04 0.00 0.00
0.23 3.20- 1.49 6.89 hCV25598595 S0028 SEX F MI_noMI G 0.12 0.08
0.0224 0.0462 0.0514 1.61 1.07- 2.41 hCV25598595 S0028 SEX F
MI_YOUNGOLD_noASD G 0.12 0.05 0.04 0.04 0.32 2.85 - 1.08 7.53
hCV25598595 S0028 HTN Y MI_noASD G 0.12 0.07 0.01 0.01 0.23 1.86
1.19 2.90- hCV25598595 S0028 HTN Y MI_noMI G 0.12 0.08 0.00 0.00
0.13 1.59 1.22 2.07 hCV25598595 S0028 HTN Y MI_YOUNGOLD_noASD G
0.12 0.04 0.01 0.01 0.28 3.16 - 1.32 7.56 hCV25598595 S0012(M) no
ALL LT60MI_GT75noMI G 0.18 0.13 0.048 1.50 1 2.2- 4 hCV25598595
S0012(M) no ALL MI_noMI G 0.17 0.13 0.020 1.39 1.05 1.82
hCV25598595 S0012(M) no ALL MI_LT75noMI G 0.17 0.13 0.033 1.39 1.03
1.87- hCV2908485 V0001 SEX M MI_noMI G 0.42 0.36 0.0348 0.0783
0.0866 1.27 1.02 - 1.59 hCV2908485 S0012(M) no ALL MI_LT75noMI G
0.45 0.38 0.0138 1.31 1.05 1.62- hCV11764545 S0012 no ALL MI_noMI T
0.21 0.18 0.07 0.05 0.61 1.22 0.99 1.50- hCV11764545 S0028 SEX F
MI_noASD T 0.22 0.15 0.03 0.03 0.32 1.60 1.05 2.45- hCV25944011
V0001 SEX M MI_noMI A 0.38 0.33 0.0392 1.3 1 1.5 hCV25944011
S0012(M) no ALL LT60MI_60TO75noMI A 0.38 0.27 0.0007 1.7 1.2- 2.2
hCV7615375 S0012 no ALL MI_noASD T 0.05 0.02 0.02 0.03 0.40 2.24
1.10 4.58- hCV7615375 S0012 no ALL MI_noMI T 0.05 0.03 0.01 0.01
0.30 1.66 1.13 2.42 hCV7615375 S0028 HTN N MI_noASD T 0.09 0.02
0.04 0.02 5.30 1.14 24.60 hCV7615375 S0028 HTN N MI_noMI T 0.09
0.02 0.03 0.01 4.46 1.32 15.09 hCV3084793 S0012 no ALL MI_noASD C
0.17 0.09 0.00 0.00 0.03 1.97 1.36 2.85- hCV3084793 S0012 no ALL
MI_YOUNGOLD_noASD C 0.17 0.09 0.00 0.00 0.05 1.97 - 1.31 2.96
hCV3084793 S0028 HTN Y MI_noASD C 0.16 0.12 0.16 0.33 0.02 1.32
0.92 1.89 hCV901792 S0012 no ALL MI_YOUNGOLD_noASD G 0.10 0.13 0.07
0.04 0.82 0.69 0- .47 1.03 hCV901792 S0012 BMI_GE27 L
MI_YOUNGOLD_noASD G 0.08 0.14 0.04 0.04 0.39 0.- 55 0.32 0.97
hCV901792 S0028 BMI_GE27 L MI_YOUNGOLD_noASD G 0.03 0.10 0.06 0.04
0.23 0- .05 1.11 hCV2188895 S0028 AGE_LT60 O MI_noMI G 0.49 0.56
0.0323 0.76 0.59 0.97 hCV2188895 S0028 AGE_LT60 O MI_noMI G 0.49
0.56 0.0323 0.76 0.59 0.97 hCV2188895 S0012(M) no ALL
LT60MI_GT75noMI G 0.52 0.60 0.0415 0.74 0.55 - 0.98 hCV2188895
S0012(M) no ALL MI_GT75noMI G 0.53 0.60 0.0468 0.75 0.57 0.98-
hCV1212713 S0012 no ALL MI_noASD G 0.46 0.35 0.02 0.06 0.05 1.56
1.08 2.26- hCV1212713 S0012 no ALL MI_noMI G 0.46 0.41 0.02 0.10
0.03 1.21 1.03 1.43 hCV1212713 S0012 BMI_GE27 L MI_noASD G 0.43
0.28 0.01 0.03 0.05 1.99 1.17 - 3.40 hCV1212713 S0012 HTN N
MI_noASD G 0.47 0.33 0.03 0.22 0.02 1.74 1.05 2.90 hCV1212713 S0028
BMI_GE27 L MI_noASD G 0.44 0.34 0.04 0.09 0.08 1.58 1.04 - 2.38
hCV1212713 S0028 HTN N MI_noMI G 0.46 0.40 0.08 0.28 0.04 1.79 0.97
3.31 hCV1212713 V0001 no ALL MI_noMI G 0.45 0.40 0.0078 0.1081
0.0041 1.24 1.06- 1.45 hCV1212713 V0001 HTN N MI_noMI G 0.46 0.41
0.0229 0.2744 0.0060 1.25 1.03 - 1.51 hCV277090 S0028 no ALL
MI_YOUNGOLD_noASD G 0.58 0.47 0.0359 1.53 1.17 1.- 97 hCV277090
S0012(F) AGE_LT60 O MI_noASD G 0.59 0.49 0.0461 1.46 1.02 2.09-
hCV2716008 S0028 no ALL MI_noMI C 0.20 0.16 0.0202 1.31 1.04 1.64
hCV2716008 S0028 no ALL MI_noASD C 0.20 0.15 0.0204 1.42 1.05 1.9
hCV2716008 S0012(M) no ALL MI_noMI C 0.24 0.19 0.0206 1.32 1.04
1.67
hCV2531086 S0028 no ALL MI_YOUNGOLD_noMI G 0.78 0.86 0.0314 0.57
0.33 0.- 95 hCV2531086 S0012(M) no ALL MI_LT75noMI G 0.77 0.82
0.0198 0.73 0.55 0.94- hCV9615318 V0001 HTN Y MI_noMI A 0.42 0.33
0.0146 0.0069 0.2786 1.46 1.08 - 1.96 hCV9615318 S0028 SMOKE N
MI_YOUNGOLD_noMI A 0.51 0.30 0.0091 2.41 1.29 4- .48 hCV9615318
S0012(M) no ALL MI_LT75noMI A 0.43 0.36 0.0128 1.32 1.06 1.63-
hCV9615318 S0012(M) no ALL MI_LT75noMI A 0.43 0.36 0.0128 1.32 1.06
1.63- hCV25602572 S0028 no ALL MI_noMI A 0.96 0.98 0.0303 0.54 0.35
0.83 hCV25602572 S0028 SMOKE Y MI_noMI A 0.97 0.99 0.0223 0.39 0.22
0.68 hCV25602572 S0012(F) SMOKE Y MI_noASD A 0.97 0.99 0.0483 0.17
0.07 0.39 hCV25603879 S0012 BMI_GE27 H MI_noASD T 0.02 0.05 0.07
0.04 0.39 0.15 1.0- 0 hCV25603879 S0028 no ALL MI_noASD T 0.01 0.17
0.16 0.03 0.07 0.00 1.38 hCV25603879 S0028 BMI_GE27 H MI_noASD T
0.02 0.25 0.14 0.01 0.06 0.00 1.1- 9 hCV1662671 S0028 no ALL
MI_noASD G 0.39 0.33 0.0440 1.27 1 1.59 hCV1662671 S0012(M) no ALL
LT60MI_GT75noMI G 0.35 0.26 0.0126 1.50 1.09 - 2.05 hCV517658 S0028
AGE_LT60 Y MI_noASD C 0.34 0.26 0.0111 1.51 1.16 1.95 hCV517658
S0012(F) no ALL YoungMI_GT75noASD C 0.39 0.28 0.0026 1.64 1.26- 2.1
hCV25608809 V0001 SMOKE Y MI_noMI A 0.55 0.61 0.0300 0.8 0.6 0.9
hCV25608809 S0012(M) SMOKE Y MI_LT75noMI A 0.52 0.60 0.0230 0.7 0.5
0.9 hCV12029981 S0028 SMOKE Y MI_noMI G 0.91 0.93 0.03942 0.69 0.48
0.98 hCV12029981 S0012(M) SMOKE Y MI_LT75noMI G 0.90 0.94 0.02876
0.55 0.32 0- .93 hCV3011239 S0028 no ALL MI_noASD A 0.39 0.33
0.0249 1.31 1.03 1.64 hCV3011239 S0012(M) no ALL LT60MI_60TO75noMI
A 0.40 0.33 0.0449 1.35 1.0- 1 1.79 hCV370782 S0012 SMOKE Y
MI_YOUNGOLD_noASD C 0.36 0.44 0.07 0.02 0.71 0.71 - 0.50 1.03
hCV370782 S0028 SMOKE Y MI_YOUNGOLD_noASD C 0.32 0.44 0.04 0.01
0.55 0.61 - 0.38 0.97 hCV25607748 S0028 SEX M MI_noASD T 0.96 0.91
0.0103 2.25 1.25 4.04 hCV25607748 S0012(M) no ALL MI_noMI T 0.94
0.92 0.0367 1.53 1.02 2.26 hCV3046056 S0028 no ALL MI_noASD G 0.96
0.93 0.0491 1.65 1.03 2.61 hCV3046056 S0012(M) no ALL MI_GT75noMI G
0.96 0.93 0.0289 1.95 1.1 3.45 hCV11758801 S0028 no ALL MI_noMI G
0.04 0.02 0.0074 2.03 1.35 3.03 hCV11758801 S0012(F) no ALL
YoungMI_GT75noASD G 0.04 0.01 0.0238 3.77 1.- 97 7.2 hCV2676035
S0028 no ALL MI_noMI G 0.63 0.59 0.0240 1.23 1.04 1.44 hCV2676035
S0028 no ALL MI_noASD G 0.63 0.58 0.0398 1.27 1.06 1.5 hCV2676035
S0012(F) no ALL MI_noASD G 0.65 0.60 0.0393 1.28 1.03 1.57
hCV16033535 S0028 SEX F MI_noMI T 0.79 0.73 0.0456 1.41 1.03 1.91
hCV16033535 S0028 SEX F MI_noASD T 0.79 0.71 0.0195 1.58 1.14 2.18
hCV16033535 S0012(F) no ALL YoungMI_GT75noASD T 0.82 0.72 0.0012
1.79 1.- 31 2.43 hCV25608687 S0028 AGE_LT60 O MI_noMI A 0.06 0.03
0.0098 2.21 1.18 4.13 hCV25608687 S0028 AGE_LT60 O MI_noASD A 0.06
0.02 0.0110 4.22 1.11 15.96- hCV25608687 S0012(M) AGE_LT60 O
MI_LT75noMI A 0.10 0.05 0.0189 2.24 1.14- 4.38 hCV9482394 S0028
SMOKE N MI_noASD A 0.12 0.07 0.04386 1.75 1.01 3.02 hCV9482394
S0012(M) SMOKE N MI_LT75noMI A 0.10 0.06 0.04581 1.91 0.98 3.- 73
hCV25610470 S0028 no ALL MI_noMI C 0.96 0.94 0.0131 1.66 1.09 2.51
hCV25610470 S0028 no ALL MI_noMI C 0.96 0.94 0.0131 1.66 1.12 2.45
hCV25610470 S0028 AGE_LT60 O MI_noASD C 0.97 0.94 0.0495 2.43 1.23
4.78 hCV25610470 S0012(F) AGE_LT60 O MI_noASD C 0.98 0.93 0.0300
3.86 1.15 12- .92 hCV25610470 S0012(M) no ALL LT60MI_GT75noMI C
0.95 0.91 0.0180 2.04 1.15- 3.59 hCV25610470 S0012(M) no ALL
MI_GT75noMI C 0.96 0.91 0.0038 2.23 1.31 3.7- 9 hCV25610773 S0028
SEX M MI_noASD G 0.11 0.06 0.0191 1.99 1.11 3.56 hCV25610773
S0012(M) no ALL MI_GT75noMI G 0.10 0.06 0.0313 1.76 1.04 2.9- 5
hCV16170641 S0028 AGE_LT60 O MI_noMI C 0.04 0.07 0.0378 0.56 0.31
0.98 hCV16170641 S0012(M) AGE_LT60 O MI_LT75noMI C 0.03 0.09 0.0126
0.34 0.13- 0.82 hCV1387523 S0012 no ALL MI_noMI A 0.17 0.22 0.00
0.01 0.03 0.73 0.59 0.90 hCV1387523 S0028 SMOKE N MI_YOUNGOLD_noASD
A 0.09 0.21 0.04 0.02 0.36 0.1- 4 0.94 hCV25610227 S0028 no ALL
MI_YOUNGOLD_noASD T 0.03 0.07 0.0220 0.33 0.15 - 0.71 hCV25610227
S0012(F) no ALL MI_noASD T 0.02 0.05 0.0490 0.50 0.27 0.93
hCV1385736 S0028 no ALL MI_noMI C 0.51 0.58 0.0005 0.73 0.61 0.87
hCV1385736 S0012(M) AGE_LT60 Y MI_LT75noMI C 0.55 0.63 0.0291 0.72
0.54 - 0.96 hCV8400671 S0028 SMOKE N MI_noASD G 0.81 0.88 0.0352
0.58 0.38 0.87 hCV8400671 S0012(F) no ALL YoungMI_GT75_noASD G 0.78
0.85 0.0278 0.64 0.- 47 0.86 hCV25614016 S0012 SEX M
MI_YOUNGOLD_noASD G 0.19 0.11 0.026 0.021 0.456 1.- 96 1.08 3.57
hCV25614016 S0012 SMOKE Y MI_YOUNGOLD_noASD G 0.19 0.11 0.029 0.011
0.900 - 1.84 1.07 3.15 hCV25614016 V0001 no ALL MI_noMI G 0.18 0.15
0.045 1.26 1.01 1.55 hCV25614016 V0001 SEX M MI_noMI G 0.18 0.14
0.019 1.44 1.06 1.95 hCV25614016 V0001 SMOKE Y MI_noMI G 0.18 0.12
0.003 1.63 1.17 2.24 hCV14938 S0028 SMOKE N MI_noMI C 0.33 0.26
0.0452 1.44 1.01 2.04 hCV14938 S0028 SMOKE N MI_noMI C 0.33 0.26
0.0452 1.44 1.04 1.98 hCV14938 S00012(F) no ALL YoungMI_GT75noASD C
0.35 0.27 0.0236 1.48 1.13- 1.91 hCV14938 S0012(M) no ALL MI_noMI C
0.29 0.24 0.0474 1.25 1 1.56 hCV8932279 S0028 SMOKE N MI_noASD G
0.49 0.39 0.02939 1.47 1.04 2.05 hCV8932279 S0028 SMOKE N MI_noMI G
0.49 0.39 0.02148 1.49 1.07 2.06 hCV8932279 S0012(M) SMOKE N
MI_LT75noMI G 0.51 0.34 0.00011 2.03 1.42 2.- 89 hCV1022614 S0028
no ALL MI_noMI A 0.16 0.21 0.0129 0.75 0.59 0.93 hCV1022614 S0028
no ALL MI_noMI A 0.16 0.21 0.0129 0.75 0.6 0.91 hCV1022614 S0012(F)
no ALL MI_noASD A 0.17 0.21 0.0500 0.75 0.57 0.98 hCV1022614
S0012(M) no ALL MI_noMI A 0.17 0.21 0.0382 0.76 0.59 0.98
hCV15851292 S0028 no ALL MI_noASD T 0.10 0.15 0.0074 0.63 0.45 0.87
hCV15851292 S0012(M) no ALL MI_GT75noMI T 0.11 0.16 0.0242 0.63
0.42 0.9- 2 hCV3068176 S0012 BMI_GE27 H MI_noMI A 0.43 0.50 0.0444
0.0524 0.1867 0.78 - 0.62 0.99 hCV3068176 S0028 AGE_LT60 O MI_noMI
C 0.60 0.52 0.0229 1.34 1.04 1.71 hCV3068176 S0028 AGE_LT60 O
MI_noASD C 0.60 0.50 0.0316 1.50 1.04 2.16 hCV3068176 S0028 no ALL
MI_noMI C 0.61 0.55 0.0042 1.28 1.07 1.5 hCV3068176 S0028 AGE_LT60
O MI_noASD C 0.62 0.52 0.00050 1.54 1.2 1.97 hCV3068176 S0028
AGE_LT60 O MI_noMI C 0.62 0.54 0.00170 1.44 1.14 1.81 hCV3068176
S0012(M) AGE_LT60 O MI_LT75noMI C 0.70 0.56 0.00149 1.81 1.25- 2.61
hCV3068176 S0012(M) AGE_LT60 O MI_LT75noMI C 0.70 0.56 0.0015 1.81
1.25 - 2.61 hCV25617360 S0028 no ALL MI_noMI T 0.16 0.19 0.0492
0.79 0.63 0.99 hCV25617360 S0012(M) no ALL LT60MI_60T075noMI T 0.15
0.21 0.0293 0.67 0.- 47 0.95 hCV795442 S0012 BMI_GE27 L
MI_YOUNGOLD_noASD G 0.29 0.33 0.30 0.05 0.30 0.- 82 0.56 1.19
hCV795442 S0028 no ALL MI_YOUNGOLD_noASD G 0.29 0.37 0.07 0.04 0.47
0.72 0- .50 1.02 hCV795442 S0028 HTN N MI_YOUNGOLD_noASD G 0.13
0.39 0.0318 0.0084 0.3331 0- .22 0.06 0.85 hCV795442 S0012(M) no
ALL LT60MI_GT75noMI G 0.14 0.20 0.023 0.65 0.44 0.- 94 hCV7490119
S0028 SEX M MI_noASD C 0.29 0.37 0.0151 0.68 0.49 0.93 hCV7490119
S0028 no ALL MI_noASD C 0.27 0.35 0.00009 0.68 0.56 0.82 hCV7490119
S0028 no ALL MI_noMI C 0.27 0.35 0.00007 0.69 0.57 0.82 hCV7490119
S0012(M) no ALL LT60MI_60TO75noMI C 0.28 0.36 0.02106 0.71 0.- 52
0.94 hCV7490119 S0012(M) no ALL LT60MI_60TO75noMI C 0.28 0.36
0.0211 0.71 0.5- 2 0.94 hCV22274307 S0028 SEX M MI_YOUNGOLD_noASD T
0.73 0.91 0.0025 0.27 0.18 0- .4 hCV22274307 S0012(F) no ALL
MI_noASD T 0.72 0.78 0.0240 0.74 0.58 0.92 hCV8827241 S0028
AGE-LT60 O MI_noMI C 0.66 0.59 0.0237 1.35 1.04 1.74 hCV8827241
V0001 no ALL MI_noMI C 0.64 0.59 0.0244 1.2 1 1.4 hCV8827241 V0001
SEX M MI_noMI C 0.64 0.58 0.0264 1.3 1 1.6 hCV8827241 S0012(M) no
ALL LT60MI_60TO75noMI C 0.67 0.59 0.0120 1.4 1 1.- 9 hCV8827241
S0012(M) no ALL LT60MI_60TO75noMI C 0.67 0.59 0.0120 1.44 1.0- 8
1.91 hCV761961 S0012 BMI_GE27 H MI_noMI T 0.28 0.21 0.00 0.01 0.02
1.51 1.15 1.- 98 hCV761961 S0012 HTN N MI_noMI T 0.28 0.23 0.07
0.27 0.01 1.32 0.99 1.77 hCV761961 S0028 no ALL MI_noASD T 0.28
0.20 0.0006 0.0013 0.0416 1.55 1.20- 2.00 hCV761961 S0028 no ALL
MI_noMI T 0.28 0.24 0.0308 0.0331 0.2243 1.21 1.02 - 1.45 hCV761961
S0028 BMI_GE27 H MI_noASD T 0.30 0.20 0.00 0.00 0.13 1.73 1.25 2-
.41 hCV761961 S0028 no ALL MI_noASD T 0.27 0.21 0.0047 1.46 1.12
1.89 hCV761961 S0012(M) no ALL MI_LT75noMI T 0.28 0.23 0.030 1.32
1.03 1.68 hCV761961 S0012(M) no ALL MI_LT75noMI T 0.28 0.23 0.0298
1.32 1.03 1.68 hCV2972952 S0012 AGE_LT60 Y MI_noASD G 0.21 0.36
0.02 0.04 0.02 0.47 0.27 - 0.84 hCV2972952 S0028 no ALL MI_noMI G
0.17 0.20 0.15 0.44 0.02 0.86 0.70 1.05 hCV2972952 S0028 AGE_LT60 Y
MI_noMI G 0.16 0.22 0.02 0.05 0.04 0.70 0.52 0- .94 hCV7538986
S0012(M) AGE_LT60 Y MI_LT75noMI A 0.06 0.03 0.0484 2.24 1.07 - 4.67
hCV7538986 S0012(M) SMOKE Y MI_LT75noMI A 0.07 0.04 0.0461 1.98
1.04 3.7- 3 hCV9514434 S0028 no ALL MI_noASD C 0.94 0.91 0.0495
1.53 1.01 2.31 hCV9514434 S0012(M) SMOKE Y MI_LT75noMI C 0.95 0.91
0.0352 1.80 1.06 3.0- 6 hCV3188386 S0012(M) no ALL MI_noMI T 0.52
0.58 0.0143 0.78 0.64 0.94 hCV3188386 S0012(M) no ALL
LT60MI_GT75noMI T 0.51 0.61 0.0071 0.67 0.5 0- .89 hCV3188386
S0012(M) AGE_LT60 Y MI_LT75noMI T 0.51 0.61 0.0039 0.66 0.49 - 0.87
hCV3188386 S0012(M) no ALL MI_GT75noMI T 0.52 0.61 0.0092 0.69 0.52
0.9 hCV1923359 S0028 no ALL MI_YOUNGOLD_noMI C 0.46 0.56 0.0368
0.66 0.45 0.- 96 hCV1923359 S0028 no ALL MI_YOUNGOLD_noASD C 0.46
0.56 0.0368 0.66 0.51 0- .85 hCV1923359 S0012(F) no ALL
YoungMI_GT75noASD C 0.41 0.51 0.0130 0.69 0.5- 3 0.87 hCV1923359
S0012(M) no ALL MI_GT75noMI C 0.50 0.57 0.0243 0.73 0.56 0.96-
hCV25623804 S0028 no ALL MI_noASD C 0.98 0.95 0.0014 2.70 1.55 4.68
hCV25623804 S0012(F) no ALL MI_noASD C 0.98 0.95 0.0138 2.36 1.24
4.5 hCV25922320 S0028 no ALL MI_noMI C 0.71 0.75 0.0472 0.82 0.68
0.97 hCV25922320 S0012(F) no ALL MI_noASD C 0.71 0.77 0.0188 0.73
0.58 0.91 hCV2259750 S0028 no ALL MI_noMI A 0.37 0.33 0.0457 1.21 1
1.44 hCV2259750 S0028 no ALL MI_noMI A 0.37 0.33 0.0457 1.21 1.02
1.42 hCV2259750 S0012(F) AGE_LT60 O MI_noASD A 0.48 0.34 0.0033
1.78 1.24 2.5- 3 hCV2259750 S0012(M) no ALL MI_GT75noMI A 0.37 0.30
0.0381 1.36 1.01 1.8 hCV1842400 S0028 SEX M MI_YOUNGOLD_noMI C 0.18
0.06 0.0296 3.54 1.17 10.- 66 hCV1842400 S0012(M) no ALL MI_noMI C
0.17 0.13 0.0169 1.39 1.06 1.82 hCV25477 S0028 SMOKE Y MI_noASD G
0.41 0.50 0.0147 0.69 0.5 0.92 hCV25477 S0012(M) SMOKE Y
MI_LT75noMI G 0.38 0.45 0.0374 0.75 0.57 0.98 hCV216064 S0012(M) no
ALL LT60MI_60TO75noMI C 0.24 0.31 0.0432 0.73 0.53- 0.98 hCV216064
S0012(M) no ALL LT60MI_60TO75noMI C 0.24 0.31 0.0432 0.73 0.53-
0.98 hCV2682687 S0012(M) AGE_LT60 Y MI_LT75noMI C 0.52 0.43 0.0120
1.43 1.08 - 1.9 hCV2682687 S0012(M) AGE_LT60 Y MI_LT75noMI C 0.52
0.43 0.0120 1.43 1.08 - 1.9 hCV3188386 S0012(M) no ALL MI_noMI T
0.52 0.58 0.0143 0.78 0.64 0.94 hCV3188386 S0012(M) no ALL
LT60MI_GT75noMI T 0.51 0.61 0.0071 0.67 0.5 0- .89 hCV3188386
S0012(M) AGE_LT60 Y MI_LT75noMI T 0.51 0.61 0.0039 0.66 0.49 - 0.87
hCV3188386 S0012(M) no ALL MI_GT75noMI T 0.52 0.61 0.0092 0.69 0.52
0.9 hCV7538986 S0012(M) AGE_LT60 Y MI_LT75noMI A 0.06 0.03 0.0484
2.24 1.07 - 4.67 hCV7538986 S0012(M) SMOKE Y MI_LT75noMI A 0.07
0.04 0.0461 1.98 1.04 3.7- 3
TABLE-US-00008 TABLE 7 Gene Marker Sample Set p-value OR* 95% CI
case_freq control_freq Allele1 mode Strata GLIO703 hCV1065191
BMS_CARE 0.016835 0.7090 0.53439 0.941 0.6102 0.6883 T Dom ALL
GLIO703 hCV1065191 BMS_PRE-CARE 0.011758 0.5837 0.38306 0.889
0.5873 0.7091 T Dom BMI_TERTILE_3 GLIO703 hCV1065191 BMS_PRE-CARE
0.036996 0.7356 0.5529 0.979 0.3947 0.4700 T Allelic
GLUCOSE_TERTILE_2 CSF2RB hCV11486078 BMS_CARE 0.045129 1.4877
1.00667 2.199 0.1535 0.1087 C Dom ALL CSF2RB hCV11486078
BMS_PRE-CARE 0.042394 1.8670 1.0146 3.435 0.1776 0.1037 C Dom
GLUCOSE_TERTILE_1 ITGAL hCV11789692 BMS_CARE 0.047325 0.5905
0.34955 0.997 0.3662 0.4946 G Dom BMI_TERTILE_1 ITGAL hCV11789692
BMS_PRE-CARE 0.012096 0.6411 0.45179 0.91 0.1984 0.2786 G Allelic
BMI_TERTILE_1 LRP2 hCV16165996 BMS_CARE 0.036433 0.1535 0.02047
1.151 0.0101 0.0623 T Rec BMI_TERTILE_3 LRP2 hCV16165996 BMS_CARE
0.011482 0.2448 0.07533 0.796 0.0197 0.0760 T Rec FMHX_CHD-0 LRP2
hCV16165996 BMS_CARE 0.041336 0.2476 0.05822 1.053 0.0168 0.0646 T
Rec HYPERTEN_1 LRP2 hCV16165996 BMS_CARE 0.049657 0.4346 0.1848
1.022 0.0272 0.0603 T Rec MALE LRP2 hCV16165996 BMS_CARE 0.021819
0.4087 0.18573 0.9 0.0276 0.0648 T Rec ALL LRP2 hCV16165996
BMS_PRE-CARE 0.045253 0.3095 0.0923 1.038 0.0233 0.0714 T Rec
AGE_TERTILE_2 LRP2 hCV16165996 BMS_PRE-CARE 0.038948 0.3702 0.13918
0.985 0.0289 0.0744 T Rec AGE_TERTILE_3 LRP2 hCV16165996
BMS_PRE-CARE 0.010388 0.1835 0.04315 0.781 0.0159 0.0808 T Rec
BMI_TERTILE_2 LRP2 hCV16165996 BMS_PRE-CARE 0.034392 0.3384 0.11833
0.968 0.0204 0.0580 T Rec PREVASTATIN LRP2 hCV16165996 BMS_PRE-CARE
0.028361 0.4124 0.18255 0.932 0.0385 0.0884 T Rec PLACEBO LRP2
hCV16165996 BMS_PRE-CARE 0.003587 0.2734 0.10784 0.693 0.0236
0.0812 T Rec FMHX_CHD-0 LRP2 hCV16165996 BMS_PRE-CARE 0.026662
0.3545 0.13642 0.921 0.0303 0.0810 T Rec HYPERTEN_1 LRP2
hCV16165996 BMS_PRE-CARE 0.036151 0.4065 0.17053 0.969 0.0282
0.0666 T Rec HYPERTEN_0 LRP2 hCV16165996 BMS_PRE-CARE 0.006692
0.4177 0.2182 0.8 0.0318 0.0729 T Rec MALE LRP2 hCV16165996
BMS_PRE-CARE 0.002522 0.3843 0.20212 0.731 0.0291 0.0724 T Rec ALL
THBS1 hCV16170900 BMS_CARE 0.018783 6.5926 1.08389 40.098 0.0357
0.0056 G Rec BMI_TERTILE_2 THBS1 hCV16170900 BMS_PRE-CARE 0.049388
1.5396 1.00678 2.354 0.1480 0.1014 G Allelic BMI_TERTILE_2 MSR1
hCV16172249 BMS_CARE 0.022431 15.7338 -- 0.0143 0.0000 C Rec
BMI_TER- TILE_1 MSR1 hCV16172249 BMS_PRE-CARE 0.026523 1.5093
1.04709 2.175 0.1303 0.0903 C Dom ALL SELL hCV16172571 BMS_CARE
0.014986 1.8087 1.11763 2.927 0.3434 0.2243 A Dom BMI_TERTILE_3
SELL hCV16172571 BMS_PRE-CARE 0.019386 2.3618 1.12523 4.957 0.0344
0.0149 A Rec ALL ADAM8 hCV16191372 BMS_CARE 0.033461 2.5166 1.0442
6.065 0.0316 0.0128 T Rec ALL ADAM8 hCV16191372 BMS_PRE-CARE
0.001246 10.0833 1.81263 56.092 0.0526 0.0055 T Rec AGE_TERTILE_1
ADAM8 hCV16191372 BMS_PRE-CARE 0.023749 1.5758 1.06064 2.341 0.3212
0.2310 T Dom HYPERTEN_1 BIRC5 hCV16266313 BMS_CARE 0.000147 3.4531
1.76512 6.755 0.1910 0.0640 G Dom AGE_TERTILE_3 BIRC5 hCV16266313
BMS_CARE 0.007451 2.2955 1.23349 4.272 0.2169 0.1077 G Dom
BMI_TERTILE_2 BIRC5 hCV16266313 BMS_CARE 0.029598 1.8335 1.05502
3.186 0.1458 0.0852 G Dom PLACEBO BIRC5 hCV16266313 BMS_CARE
0.033243 1.9616 1.04542 3.681 0.1584 0.0876 G Dom FMHX_CHD-1 BIRC5
hCV16266313 BMS_CARE 0.038827 2.1011 1.0259 4.303 0.1494 0.0772 G
Dom GLUCOSE_TERTILE_1 BIRC5 hCV16266313 BMS_CARE 0.005253 2.6022
1.3547 4.998 0.0792 0.0320 G Allelic GLUCOSE_TERTILE_3 BIRC5
hCV16266313 BMS_CARE 0.027848 1.6231 1.05066 2.507 0.1448 0.0945 G
Dom MALE BIRC5 hCV16266313 BMS_CARE 0.026078 1.6010 1.05444 2.431
0.1344 0.0884 G Dom ALL BIRC5 hCV16266313 BMS_PRE-CARE 0.006131
2.0933 1.26582 3.462 0.1085 0.0550 G Allelic AGE_TERTILE_2 BIRC5
hCV16266313 BMS_PRE-CARE 0.014989 1.9766 1.15433 3.385 0.0952
0.0506 G Allelic BMI_TERTILE_1 BIRC5 hCV16266313 BMS_PRE-CARE
0.00485 2.0006 1.22638 3.263 0.1703 0.0931 G Dom PLACEBO BIRC5
hCV16266313 BMS_PRE-CARE 0.021465 1.6815 1.07589 2.628 0.1604
0.1020 G Dom FMHX_CHD-0 BIRC5 hCV16266313 BMS_PRE-CARE 0.010177
2.1607 1.18862 3.928 0.1963 0.1015 G Dom GLUCOSE_TERTILE_1 BIRC5
hCV16266313 BMS_PRE-CARE 0.03938 1.8321 1.0367 3.238 0.0790 0.0447
G Allelic GLUCOSE_TERTILE_2 BIRC5 hCV16266313 BMS_PRE-CARE 0.00124
4.2532 1.68033 10.766 0.3125 0.0966 G Dom FEMALE BIRC5 hCV16266313
BMS_PRE-CARE 0.014904 1.5129 1.08876 2.102 0.0767 0.0521 G Allelic
ALL F13A1 hCV1639938 BMS_CARE 0.034558 0.6743 0.47218 0.963 0.2018
0.2727 A Allelic PREVASTATIN F13A1 hCV1639938 BMS_PRE-CARE 0.012496
0.6988 0.52904 0.923 0.2041 0.2684 A Allelic PREVASTATIN PDGFRA
hCV22271841 BMS_CARE 0.001 9.6964 1.86162 50.505 0.0345 0.0037 C
Rec PLACEBO PDGFRA hCV22271841 BMS_CARE 0.020901 3.2843 1.12941
9.551 0.0236 0.0073 C Rec ALL PDGFRA hCV22271841 BMS_PRE-CARE
0.026695 8.5140 0.87997 82.375 0.0165 0.0020 C Rec PLACEBO PDGFRA
hCV22271841 BMS_PRE-CARE 0.032784 13.8571 -- 0.0313 0.0000 C Rec F-
EMALE PON1 hCV2259750 BMS_CARE 0.040187 2.7282 1.01487 7.334 0.2121
0.0898 T Rec FEMALE PON1 hCV2259750 BMS_PRE-CARE 0.016576 3.0588
1.18628 7.887 0.8125 0.5862 T Dom FEMALE NPC1 hCV25472673 BMS_CARE
0.008339 1.4341 1.10402 1.863 0.4722 0.3842 C Allelic PLACEBO NPC1
hCV25472673 BMS_PRE-CARE 0.032505 1.6625 1.03965 2.659 0.1758
0.1137 C Rec PLACEBO ICAM3 hCV25473653 BMS_CARE 0.029271 0.5809
0.35844 0.942 0.1447 0.2256 C Allelic AGE_TERTILE_1 ICAM3
hCV25473653 BMS_CARE 0.027136 0.5830 0.36006 0.944 0.2772 0.3968 C
Dom GLUCOSE_TERTILE_3 ICAM3 hCV25473653 BMS_PRE-CARE 0.033872
0.2336 0.05434 1.004 0.0147 0.0601 C Rec GLUCOSE_TERTILE_3 ICAM3
hCV25473653 BMS_PRE-CARE 0.002233 0.3106 0.14087 0.685 0.0186
0.0576 C Rec ALL SELL hCV25474627 BMS_CARE 0.014986 1.8087 1.11763
2.927 0.3434 0.2243 A Dom BMI_TERTILE_3 SELL hCV25474627
BMS_PRE-CARE 0.018572 2.3750 1.1315 4.985 0.0345 0.0148 A Rec ALL
HADHSC hCV25594697 BMS_CARE 0.035834 0.6369 0.41669 0.973 0.1102
0.1629 C Dom ALL HADHSC hCV25594697 BMS_PRE-CARE 0.044643 0.5487
0.3033 0.992 0.0904 0.1533 C Dom FMHX_CHD-1 LRP3 hCV25594815
BMS_CARE 0.028825 5.9294 0.97529 36.049 0.0341 0.0059 T Rec
AGE_TERTILE_2 LRP3 hCV25594815 BMS_CARE 0.004599 3.3092 1.37903
7.941 0.0354 0.0110 T Rec ALL LRP3 hCV25594815 BMS_PRE-CARE
0.021685 9.0480 0.93272 87.771 0.0234 0.0027 T Rec AGE_TERTILE_2
LRP3 hCV25594815 BMS_PRE-CARE 0.002526 8.1321 1.62452 40.708 0.0364
0.0046 T Rec HYPERTEN_1 LRP3 hCV25594815 BMS_PRE-CARE 0.027417
9.6000 0.84307 109.315 0.0625 0.0069 T Rec FEMALE CR1 hCV25596020
BMS_CARE 0.011753 2.9864 1.22737 7.266 0.0756 0.0267 G Rec
HYPERTEN_1 CR1 hCV25596020 BMS_PRE-CARE 0.049789 2.4429 0.97421
6.126 0.0552 0.0234 G Rec HYPERTEN_1 SLC21A3 hCV25605897 BMS_CARE
0.011574 0.3192 0.12582 0.81 0.0459 0.1309 G Dom PREVASTATIN
SLC21A3 hCV25605897 BMS_PRE-CARE 0.002728 0.3812 0.19484 0.746
0.0255 0.0643 G Allelic PREVASTATIN SLC21A3 hCV25605897
BMS_PRE-CARE 0.000185 0.4370 0.27647 0.691 0.0291 0.0642 G Allelic
ALL TLR2 hCV25607736 BMS_CARE 0.030239 2.3361 1.06404 5.129 0.1409
0.0656 A Dom BMI_TERTILE_1 TLR2 hCV25607736 BMS_PRE-CARE 0.00334
20.1963 -- 0.0080 0.0000 A Rec ALL PROCR hCV25620145 BMS_CARE
0.048224 7.8372 0.70242 87.443 0.0227 0.0030 G Rec AGE_TERTILE_2
PROCR hCV25620145 BMS_CARE 0.004479 13.0000 1.33617 126.481 0.0333
0.0027 G Rec AGE_TERTILE_3 PROCR hCV25620145 BMS_CARE 0.005918
7.7474 1.39802 42.933 0.0404 0.0054 G Rec BMI_TERTILE_3 PROCR
hCV25620145 BMS_CARE 0.048924 4.4027 0.8799 22.029 0.0197 0.0046 G
Rec FMHX_CHD-0 PROCR hCV25620145 BMS_CARE 0.007748 11.6020 1.1938
112.756 0.0297 0.0026 G Rec GLUCOSE_TERTILE_3 PROCR hCV25620145
BMS_CARE 0.008012 1.8020 1.18196 2.747 0.1513 0.0900 G Allelic
HYPERTEN_1 PROCR hCV25620145 BMS_CARE 0.001821 7.1451 1.69457
30.127 0.0226 0.0032 G Rec MALE PROCR hCV25620145 BMS_CARE 0.01132
4.3815 1.25884 15.25 0.0197 0.0046 G Rec ALL PROCR hCV25620145
BMS_PRE-CARE 0.015417 14.8039 -- 0.0155 0.0000 G Rec AG-
E_TERTILE_2 PROCR hCV25620145 BMS_PRE-CARE 0.005072 17.9204 --
0.0231 0.0000 G Rec AG- E_TERTILE_3 PROCR hCV25620145 BMS_PRE-CARE
0.016453 14.5181 -- 0.0159 0.0000 G Rec BM- I_TERTILE_3 PROCR
hCV25620145 BMS_PRE-CARE 0.024768 8.7317 0.89991 84.722 0.0238
0.0028 G Rec BMI_TERTILE_2 PROCR hCV25620145 BMS_PRE-CARE 0.001456
14.3503 1.66518 123.668 0.0275 0.0020 G Rec PLACEBO PROCR
hCV25620145 BMS_PRE-CARE 0.002509 13.1366 1.52306 113.306 0.0301
0.0024 G Rec FMHX_CHD-1 PROCR hCV25620145 BMS_PRE-CARE 0.003635
20.0112 -- 0.0219 0.0000 G Rec GL- UCOSE_TERTILE_3 PROCR
hCV25620145 BMS_PRE-CARE 0.002342 22.0048 -- 0.0280 0.0000 G Rec
GL- UCOSE_TERTILE_1 PROCR hCV25620145 BMS_PRE-CARE 0.001181 24.0464
-- 0.0242 0.0000 G Rec HY- PERTEN_1 PROCR hCV25620145 BMS_PRE-CARE
0.000459 16.4471 1.97291 137.11 0.0173 0.0011 G Rec MALE PROCR
hCV25620145 BMS_PRE-CARE 0.000376 10.1509 2.09945 49.08 0.0185
0.0019 G Rec ALL TAP1 hCV25630686 BMS_CARE 0.04021 12.6872 --
0.0111 0.0000 T Rec AGE_TERT- ILE_3 TAP1 hCV25630686 BMS_CARE
0.000322 30.4354 -- 0.0118 0.0000 T Rec ALL TAP1 hCV25630686
BMS_PRE-CARE 0.018347 2.6736 1.14742 6.23 0.0756 0.0297 T Dom
AGE_TERTILE_3 LRP2 hCV25646248 BMS_CARE 0.024691 5.3000 1.04828
26.796 0.0411 0.0080 T Dom AGE_TERTILE_1 LRP2 hCV25646248
BMS_PRE-CARE 0.022547 3.6575 1.25034 10.699 0.0395 0.0111 T Allelic
AGE_TERTILE_1 HLA-DPB1 hCV25651174 BMS_CARE 0.029542 1.4094 1.03931
1.911 0.3670 0.2914 G Allelic PREVASTATIN HLA-DPB1 hCV25651174
BMS_PRE-CARE 0.036904 1.4164 1.02066 1.965 0.5714 0.4849 G Dom
PREVASTATIN NEUROD1 hCV25651593 BMS_CARE 0.046449 0.1212 -- 0.0000
0.0500 T Dom AGE_T- ERTILE_1 NEUROD1 hCV25651593 BMS_PRE-CARE
0.04905 0.5689 0.32222 1.004 0.0397 0.0677 T Dom ALL PON2
hCV2630153 BMS_CARE 0.023524 1.5374 1.05786 2.234 0.4925 0.3870 C
Dom HYPERTEN_0 PON2 hCV2630153 BMS_PRE-CARE 0.0491 1.2936 1.00802
1.66 0.2747 0.2264 C Allelic HYPERTEN_0 ABCA1 hCV2741104 BMS_CARE
0.032615 0.6617 0.45235 0.968 0.3517 0.4506 T Dom PLACEBO ABCA1
hCV2741104 BMS_CARE 0.019416 0.5838 0.37069 0.919 0.3564 0.4868 T
Dom GLUCOSE_TERTILE_3 ABCA1 hCV2741104 BMS_PRE-CARE 0.000279 0.5035
0.34649 0.732 0.2637 0.4157 T Dom PLACEBO ABCA1 hCV2741104
BMS_PRE-CARE 0.02491 0.7932 0.64814 0.971 0.2050 0.2454 T Allelic
ALL PECAM1 hCV435368 BMS_CARE 0.007531 1.6056 1.14496 2.252 0.4464
0.4358 A Allelic BMI_TERTILE_2 PECAM1 hCV435368 BMS_CARE 0.014604
1.7845 1.11621 2.853 0.2844 0.1822 A Rec PREVASTATIN PECAM1
hCV435368 BMS_CARE 0.006582 1.5923 1.13818 2.228 0.4318 0.4525 A
Allelic GLUCOSE_TERTILE_1 PECAM1 hCV435368 BMS_CARE 0.007332 1.4384
1.10501 1.872 0.4482 0.4612 A Allelic HYPERTEN_0 PECAM1 hCV435368
BMS_CARE 0.022746 1.2736 1.03468 1.568 0.4774 0.4622 A Allelic MALE
PECAM1 hCV435368 BMS_CARE 0.017633 1.2686 1.04533 1.54 0.4822
0.4584 A Allelic ALL PECAM1 hCV435368 BMS_PRE-CARE 0.000689 1.6513
1.23632 2.206 0.4600 0.4155 A Allelic BMI_TERTILE_3 PECAM1
hCV435368 BMS_PRE-CARE 0.027008 1.3129 1.03237 1.67 0.5000 0.4324 A
Allelic PLACEBO PECAM1 hCV435368 BMS_PRE-CARE 0.0286 1.2842 1.03084
1.6 0.4858 0.4518 A Allelic FMHX_CHD-0 PECAM1 hCV435368
BMS_PRE-CARE 0.000544 1.6350 1.23863 2.158 0.4453 0.4325 A Allelic
GLUCOSE_TERTILE_3 PECAM1 hCV435368 BMS_PRE-CARE 0.011773 1.3314
1.06914 1.658 0.4742 0.4544 A Alielic HYPERTEN_0 PECAM1 hCV435368
BMS_PRE-CARE 0.010962 1.2560 1.05449 1.496 0.4913 0.4519 A Allelic
MALE
PECAM1 hCV435368 BMS_PRE-CARE 0.013871 1.2353 1.04629 1.458 0.4974
0.4500 A Allelic ALL A2M hCV517658 BMS_CARE 0.028082 1.7449 1.05782
2.878 0.6627 0.5296 G Dom BMI_TERTILE_2 A2M hCV517658 BMS_CARE
0.027305 1.3755 1.03564 1.827 0.6375 0.5611 G Dom ALL A2M hCV517658
BMS_PRE-CARE 0.007777 1.7717 1.15969 2.707 0.6532 0.5153 G Dom
BMI_TERTILE_2 A2M hCV517658 BMS_PRE-CARE 0.026334 1.7745 1.06464
2.958 0.1718 0.1047 G Rec HYPERTEN_1 ADAMTS1 hCV529706 BMS_CARE
0.029249 1.6701 1.05032 2.655 0.5667 0.4392 C Dom AGE_TERTILE_3
ADAMTS1 hCV529706 BMS_PRE-CARE 0.02017 1.6853 1.08049 2.629 0.0878
0.0540 C Rec ALL ADAMTS1 hCV529710 BMS_CARE 0.029249 1.6701 1.05032
2.655 0.5667 0.4392 C Dom AGE_TERTILE_3 ADAMTS1 hCV529710
BMS_PRE-CARE 0.016984 1.7117 1.09605 2.673 0.0873 0.0529 C Rec ALL
HLA-G hCV7482175 BMS_CARE 0.018628 0.5823 0.3697 0.917 0.2015
0.3023 A Dom HYPERTEN_0 HLA-G hCV7482175 BMS_PRE-CARE 0.037108
0.1101 -- 0.0000 0.0201 A Rec HYPE- RTEN_0 LYZ hCV7559757 BMS_CARE
0.024609 15.2416 -- 0.0133 0.0000 A Rec AGE_TERTI- LE_1 LYZ
hCV7559757 BMS_CARE 0.037787 12.9802 -- 0.0066 0.0000 A Rec
FMHX_CHD-- 0 LYZ hCV7559757 BMS_CARE 0.003322 21.6600 -- 0.0079
0.0000 A Rec ALL LYZ hCV7559757 BMS_PRE-CARE 0.022269 9.9178
0.88758 110.821 0.0267 0.0028 A Rec AGE_TERTILE_1 FABP2 hCV761961
BMS_CARE 0.036056 0.5833 0.35113 0.969 0.3684 0.5000 T Dom
AGE_TERTILE_1 FABP2 hCV761961 BMS_PRE-CARE 0.036725 0.5833 0.35052
0.971 0.3684 0.5000 T Dom AGE_TERTILE_1 MMRN hCV8933098 BMS_CARE
0.04082 1.9109 1.01944 3.582 0.1954 0.1128 G Dom AGE_TERTILE_2 MMRN
hCV8933098 BMS_PRE-CARE 0.009857 2.0313 1.17676 3.506 0.1938 0.1058
G Dom AGE_TERTILE_2 PON2 hCV8952817 BMS_CARE 0.024507 1.5312
1.05461 2.223 0.4889 0.3845 C Dom HYPERTEN_0 PON2 hCV8952817
BMS_PRE-CARE 0.04204 1.2993 1.0124 1.668 0.2747 0.2257 C Allelic
HYPERTEN_0 MC3R hCV9485713 BMS_CARE 0.016123 1.9962 1.12843 3.531
0.2529 0.1450 A Dom AGE_TERTILE_2 MC3R hCV9485713 BMS_CARE 0.020971
1.4944 1.06081 2.105 0.2143 0.1543 A Dom ALL MC3R hCV9485713
BMS_PRE-CARE 0.014872 1.9047 1.12737 3.218 0.2093 0.1220 A Dom
AGE_TERTILE_2 *Haldane OR was used if either case count or the
control count is zero
SEQUENCE LISTINGS
0 SQTB SEQUENCE LISTING The patent contains a lengthy "Sequence
Listing" section. A copy of the "Sequence Listing" is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US07482117B2)-
. An electronic copy of the "Sequence Listing" will also be
available from the USPTO upon request and payment of the fee set
forth in 37 CFR 1.19(b)(3).
* * * * *
References